US12442815B1

US12442815B1 - Viscoelastic hydrogel regulation of organoid patterning and vascularization

Info

Publication number: US12442815B1
Application number: US18/952,423
Authority: US
Inventors: Changchun Zeng; Yan Li; Xingchi Chen
Original assignee: Florida State University Research Foundation Inc
Current assignee: Florida State University Research Foundation Inc
Priority date: 2024-09-20
Filing date: 2024-11-19
Publication date: 2025-10-14
Anticipated expiration: 2044-11-19

Abstract

Disclosed herein are spinal cord models including a cell scaffold including methacrylated hyaluronic acid (HAMA) and dopamine-modified hyaluronic acid (HA-Cat), and a spinal cord spheroid or organoid or a fragment thereof cultured on the cell scaffold. Said spinal cord spheroids or organoids or fragments thereof can be co-cultured with blood vessel spheroid organoids to form blood-spinal cord barrier models. Also disclosed herein are methods of making and using said models.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Application No. 63/697,111, filed Sep. 20, 2024, which is incorporated by reference herein in its entirety.

GOVERNMENT SUPPORT CLAUSE

This invention was made with government support under Grant No. 1917618 awarded by the National Science Foundation. The Government has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING

The sequence listing submitted on Nov. 19, 2024, as an .XML file entitled “10850-118US1_ST26.xml” created on Nov. 14, 2024, and having a file size of 51,882 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

BACKGROUND

The spinal cord is part of the central nervous system and provides a connection between the brain and lower back, which delivers nerve signals from the brain to the body to control locomotion and feeling sensations [1]. Human induced pluripotent stem cells (hiPSCs) can be directly induced into different types of region-specific brain organoids, including spinal cord organoids, for studying neurodevelopment and neurodegeneration [2]. These three-dimensional (3D) organoids are usually generated in suspension. To generate different subtypes of neuronal cells in vitro such as motor neurons, hiPSCs can be induced by small molecules to become functional neural cells with a high conversion rate in a 2D culture [3], and these cells can be assembled into 3D neural structure [4]. Currently, there are still many limitations for developing more complex systems in 3D organoids. For example, the lack of specific mature pattern structure, such as rostro-caudal patterning, decreased disease modeling accuracy and reduced model effectiveness [5]. Furthermore, small molecules may not be sufficient to provide spatial cues for specific cells arranged in 3D structure, which are essential for functional neuronal and synapse maturation. Additionally, environmental stimulations, such as chemical and mechanical cues, could be less effective in 3D organoids compared to in vivo environment due to the missing signaling in vitro, which may lead to the lack of function [6, 7].

Therefore, methods with more in vivo-like microenvironment are needed to pattern hiPSC-derived spinal cord organoids, and to provide new insights into the principles of tissue patterning during spinal neurogenesis [8]. These needs and others are at least partially satisfied by the present disclosure.

SUMMARY

In one aspect, provided is a spinal cord model including: a cell scaffold including methacrylated hyaluronic acid (HAMA) and dopamine-modified hyaluronic acid (HA-Cat); and a spinal cord spheroid or organoid or a fragment thereof cultured on the cell scaffold and expressing one or more ventral markers, dorsal markers, and/or interneuron markers.

In another aspect, provided is a blood-spinal cord barrier model including a spinal cord spheroid or organoid or a fragment thereof derived from any of the disclosed spinal cord models co-cultured with a blood vessel spheroid or organoid or a fragment thereof.

In yet another aspect, provided is a method of modeling a spinal cord, the method including culturing a plurality of induced pluripotent stem cells on a cell scaffold to form a spinal cord spheroid or organoid or a fragment thereof, the cell scaffold including methacrylated hyaluronic acid (HAMA) and dopamine-modified hyaluronic acid (HA-Cat); wherein the method causes the spinal cord spheroid or organoid or a fragment thereof to express one or more ventral markers, dorsal markers, and/or interneuron markers.

In yet still another aspect, provided is a method of modeling a blood-spinal cord barrier, the method including: a) culturing a plurality of induced pluripotent stem cells on a cell scaffold to form a spinal cord spheroid or organoid or a fragment thereof, the cell scaffold including methacrylated hyaluronic acid (HAMA) and dopamine-modified hyaluronic acid (HA-Cat); and b) co-culturing the spinal cord spheroid or organoid or a fragment thereof with a blood vessel spheroid or organoid or a fragment thereof; wherein step a) causes the spinal cord spheroid or organoid or a fragment thereof to express one or more ventral markers, dorsal markers, and/or interneuron markers.

In yet still another aspect, provided is a method of screening a therapeutic agent, the method including: a) culturing a plurality of induced pluripotent stem cells on a cell scaffold to form a spinal cord spheroid or organoid or a fragment thereof, the cell scaffold including methacrylated hyaluronic acid (HAMA) and dopamine-modified hyaluronic acid (HA-Cat); and b) administering the therapeutic agent to the spinal cord spheroid or organoid or fragment thereof.

In yet still another aspect, provided is a method of screening a therapeutic agent, the method including: a) culturing a plurality of induced pluripotent stem cells on a cell scaffold to form a spinal cord spheroid or organoid or a fragment thereof, the cell scaffold including methacrylated hyaluronic acid (HAMA) and dopamine-modified hyaluronic acid (HA-Cat); b) culturing a plurality of induced pluripotent stem cells on the cell scaffold to form a spinal cord spheroid or organoid or a fragment thereof; c) co-culturing the spinal cord spheroid or organoid or a fragment thereof with a blood vessel spheroid or organoid or a fragment thereof; and d) administering the therapeutic agent to the spinal cord spheroid or organoid or fragment thereof and/or the blood vessel spheroid or organoid or a fragment thereof.

Other systems, methods, features and/or advantages will be or may become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features and/or advantages be included within this description and be protected by the accompanying claims.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1D depict HAMA synthesis and characterization. FIG. 1A is a schematic illustration of methods of (i) fabrication and synthesis of HAMA, (ii) fabrication and synthesis of HAMA@HA-Cat, (iii) fabrication and synthesis of hydrogels, and (iv) Fe³⁺curing HA-Cat hydrogels. FIG. 1B shows quantification of the compression modulus, and FIG. 1C shows tan δ of the hydrogels by compression and rheological test. (n>3 measurements per gel). FIG. 1D shows the stress relaxation test applied to the 4 selected HAMA hydrogels and regression performed by a modified Maxwell model to get stress relaxation time.

FIGS. 2A-2D depict characterizations of HAMA hydrogels. FIG. 2A is ¹H NMR results which show the success of grafting MA group to HA chains. FIG. 2B shows fabricating the HAMA library with different MW and concentrations. FIGS. 2C-2D show four types of HAMA hydrogels were selected from a series of HAMA hydrogels with different concentrations to make a library for matching sample properties. FIG. 2C shows a plot of viscoelasticity (tan δ) of all HAMA hydrogels. All tan δ values were obtained under 1 Hz. FIG. 2D shows a plot of compression modulus of all HAMA hydrogels.

FIG. 3 depicts quantification of the compression modulus for HAMA hydrogels versus strain. N=4 independent gels for each composition.

FIGS. 4A-4C depict frequency sweep of different HAMA hydrogels: 100 k (FIG. 4A), 200 k (FIG. 4B), and 1000 k (FIG. 4C).

FIG. 5 depicts scanning electron microcopy (SEM) images for freeze-dried HAMA hydrogels with different concentrations.

FIGS. 6A-6E depict ventral hSCO differentiation and characterization. FIG. 6A is a schematic illustration of ventral hSCO differentiation protocol. FIGS. 6B-6D show immunostaining and flow cytometry analysis for marker expression of hSCO differentiation. FIG. 6B and FIG. 6D were taken using confocal microscopy. Scale bar=50 μm. FIG. 6E shows quantitative RT-PCR for relative mRNA expression of (i) ventral markers, (ii) interneuron markers and (iii) dorsal markers after biochemical induction (n=3). * indicates p≤0.05, **: p≤0.01, ***: p≤0.001.

FIGS. 7A-7B depict RT-PCR analysis for hSCO differentiation. FIG. 7A shows a comparison of the differentiation markers between iPSC aggregates and hSCO spheroids. FIG. 7B shows a ventral hSCO marker expression in the organoids derived from different differentiation methods. * indicates p≤0.05, **: p≤0.01, ***: p≤0.001.

FIGS. 8A-8B depict images of hSCOs after 40 days culture. FIG. 8A shows hSCO at day 40 with static culture. scale bar=200 m. FIG. 8B shows replated day-44 hSCO with axons extensions. Scale bar=50 m.

FIG. 9 depicts immunostaining images of ventral hSCO markers. Scale bar: 50 μm.

FIGS. 10A-10E depict biocompatibility of the HAMA hydrogels and morphogenesis of the organoids. FIG. 10A shows hiPSC culture with HAMA and HAMA/Matrigel mixture for 7 days. Scale bar=50 μm. FIG. 10B shows a DNA assay, and FIG. 10C shows Live/Dead flow cytometry analysis for determining proliferation rate and survival rate of hiPSCs cultured with different HAMA hydrogels, respectively. FIG. 10D shows images of morphology of the organoids with different hydrogels over the time. Scale bar=200 μm. FIG. 10E shows quantification of diameter and circularity of hSCOs cultured in different HAMA hydrogels for morphogenesis. * indicates p≤0.05, **: p≤0.01, ***: p≤0.001.

FIG. 11 depicts hSCO morphogenesis during the induction period in different hydrogels. Scale bar: 200 μm.

FIGS. 12A-12D depict characterization for differentiation of hSCOs in different HAMA hydrogels. FIG. 12A shows flow cytometry analysis of expression of different ventral markers when generating hSCOs in different hydrogels. FIG. 12B shows a summary of 3 runs of flow cytometry analysis for identification ventral hSCO marker expression. FIG. 12C shows RT-PCR analysis of relative mRNA expression for different region-specific patterning markers during generation of hSCOs at day 35 (n=3). * indicates p≤0.05, **: p≤0.01, ***: p≤0.001. FIG. 12D shows (i) electrophysiology to show sodium and potassium currents for the replated hSCOs at day 40, and (ii) morphology of outgrowth cells of the replated hSCOs for electrophysiology. Scale bar=20 μm.

FIG. 13 depicts flow cytometry analysis of different markers for hSCO formed in different hydrogels. For each histogram, X-axis: Alex 488 intensity of marker of interest; Y-axis: relative frequency.

FIGS. 14A-14B depict endothelial markers expression by different culture condition for hBVOs. FIG. 14A shows EC marker expression of hBVOs derived from different seeding densities (10 k, 20 k, 30 k), replating condition (re), and protocols (hBVO vs. vsc protocol). FIG. 14B shows EC marker expression of hBVOs from the differentiation by adding different growth factors BMP4 and VEGF.

FIGS. 15A-15C depict hSCO and hBVO coculturing for Blood-Spinal Cord Barrier (BSCB) generation. FIG. 15A shows morphology of the merging process of two types of organoids indicated by cell-tracker (red) hBVOs. FIGS. 15B-15C show RT-PCR analysis for relative mRNA expression of ventral spinal cord genes, endothelial cells (EC), and blood-brain barrier (BBB) genes during hBVO and different hSCO coculturing. n=3, ns: p>0.05, * indicates p≤0.05, **: p≤0.01, ***: p≤0.001.

FIGS. 16A-16E depict fabrication and characterization of HAMA@HA-Cat hydrogels. The dynamic hydrogels were fabricated to enhance the hydrogel properties and potential ability to regulate hSCO derivation. FIGS. 16A-16E show rheological test and compression test performed to determine mechanical properties for the four hydrogels (Gel 5-8 in sequence). FIG. 16A shows storage modulus, FIG. 16B shows tan δ, FIG. 16C shows compression modulus, and FIG. 16D shows the viscoelasticity of the hydrogels further determined by stress relaxation test. FIG. 16E shows RT-PCR analysis of relative mRNA expression for different region-specific patterning markers of hSCOs at day 35. n=3, * indicates p≤0.05.

FIGS. 17A-17B depict histological sections for YAP localization to reveal the mechanism of hydrogel effects on hSCO patterning. FIG. 17A shows images of YAP localization. Scale bar: 50 μm. FIG. 17B shows the quantitative measurements of nuclear to cytoplasmic YAP localization for different hydrogel conditions. * indicates p≤0.05, **: p≤0.01.

DETAILED DESCRIPTION

It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate aspects, can also be provided in combination with a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, can also be provided separately or in any suitable subcombination. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure.

Definitions

In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:

As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of” and “consisting of.” Similarly, the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.

As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound”, “a composition”, or “a cancer”, includes, but is not limited to, two or more such compounds, compositions, or cancers, and the like.

It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It can be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it can be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

When a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less' and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

As used herein, the term “effective amount” refers to an amount that is sufficient to achieve the desired modification of a physical property of the composition or material. For example, an “effective amount” of a monomer refers to an amount that is sufficient to achieve the desired improvement in the property modulated by the formulation component, e.g. desired antioxidant release rate or viscoelasticity. The specific level in terms of wt % in a composition required as an effective amount will depend upon a variety of factors including the amount and type of monomer, amount and type of polymer, e.g., acrylamide, amount of antioxidant, and desired release kinetics.

As used herein, the term “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors within the knowledge and expertise of the health practitioner and which may be well known in the medical arts. In the case of treating a particular disease or condition, in some instances, the desired response can be inhibiting the progression of the disease or condition. This may involve only slowing the progression of the disease temporarily. However, in other instances, it may be desirable to halt the progression of the disease permanently. This can be monitored by routine diagnostic methods known to one of ordinary skill in the art for any particular disease. The desired response to treatment of the disease or condition also can be delaying the onset or even preventing the onset of the disease or condition.

For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. It is generally preferred that a maximum dose of the pharmacological agents of the invention (alone or in combination with other therapeutic agents) be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.

A response to a therapeutically effective dose of a disclosed drug delivery composition can be measured by determining the physiological effects of the treatment or medication, such as the decrease or lack of disease symptoms following administration of the treatment or pharmacological agent. Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response. The amount of a treatment may be varied for example by increasing or decreasing the amount of a disclosed compound and/or pharmaceutical composition, by changing the disclosed compound and/or pharmaceutical composition administered, by changing the route of administration, by changing the dosage timing and so on. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.

As used herein, the term “prophylactically effective amount” refers to an amount effective for preventing onset or initiation of a disease or condition.

As used herein, the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.

As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

As used interchangeably herein, “subject,” “individual,” or “patient” can refer to a vertebrate organism, such as a mammal (e.g. human). “Subject” can also refer to a cell, a population of cells, a tissue, an organ, or an organism, preferably to human and constituents thereof.

As used herein, the terms “treating” and “treatment” can refer generally to obtaining a desired pharmacological and/or physiological effect. The effect can be, but does not necessarily have to be, prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof. The effect can be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease, disorder, or condition. The term “treatment” as used herein can include any treatment of a disease disorder in a subject, particularly a human and can include any one or more of the following: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., mitigating or ameliorating the disease and/or its symptoms or conditions. The term “treatment” as used herein can refer to both therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. Those in need of treatment (subjects in need thereof) can include those already with the disorder and/or those in which the disorder is to be prevented. As used herein, the term “treating”, can include inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and/or condition. Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, e.g., such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.

As used herein, “dose,” “unit dose,” or “dosage” can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of a disclosed compound and/or a pharmaceutical composition thereof calculated to produce the desired response or responses in association with its administration.

As used herein, “therapeutic” can refer to treating, healing, and/or ameliorating a disease, disorder, condition, or side effect, or to decreasing in the rate of advancement of a disease, disorder, condition, or side effect.

Spinal Cord Model

In some aspects, the cell scaffold can include up to about 2 wt % HAMA (e.g., up to about 1.9 wt %, up to about 1.8 wt %, up to about 1.7 wt %, up to about 1.6 wt %, up to about 1.5 wt %, up to about 1.4 wt %, up to about 1.3 wt %, up to about 1.2 wt %, up to about 1.1 wt %, up to about 1 wt %, up to about 0.9 wt %, up to about 0.8 wt %, up to about 0.7 wt %, up to about 0.6 wt %, up to about 0.5 wt %, up to about 0.4 wt %, up to about 0.3 wt %, up to about 0.2 wt %, up to about 0.1 wt %, 0 wt %). In some aspects, the cell scaffold can include greater than 0 wt % HAMA (e.g., greater than about 0.1 wt %, greater than about 0.2 wt %, greater than about 0.3 wt %, greater than about 0.4 wt %, greater than about 0.5 wt %, greater than about 0.6 wt %, greater than about 0.7 wt %, greater than about 0.8 wt %, greater than about 0.9 wt %, greater than about 1 wt %, greater than about 1.1 wt %, greater than about 1.2 wt %, greater than about 1.3 wt %, greater than about 1.4 wt %, greater than about 1.5 wt %, greater than about 1.6 wt %, greater than about 1.7 wt %, greater than about 1.8 wt %, greater than about 1.9 wt %, greater than about 2 wt %).

It is considered that the cell scaffold can include an amount of HAMA ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the cell scaffold can include from 0 wt % to about 2 wt % HAMA (e.g., from about 0.1 wt % to about 1.9 wt %, from about 0.2 wt % to about 1.8 wt %, from about 0.3 wt % to about 1.7 wt %, from about 0.4 wt % to about 1.6 wt %, from about 0.5 wt % to about 1.5 wt %, from about 0.6 wt % to about 1.4 wt %, from about 0.7 wt % to about 1.3 wt %, from about 0.8 wt % to about 1.2 wt %, from about 0.9 wt % to about 1.1 wt %, from 0 wt % to about 1 wt %, from about 0.1 wt % to about 0.9 wt %, from about 0.2 wt % to about 0.8 wt %, from about 0.3 wt % to about 0.7 wt %, from about 0.4 wt % to about 0.6 wt %, from about 1 wt % to about 2 wt %, from about 1.1 wt % to about 1.9 wt %, from about 1.2 wt % to about 1.8 wt %, from about 1.3 wt % to about 1.7 wt %, from about 1.4 wt % to about 1.6 wt %).

In some aspects, the cell scaffold can include up to about 2 wt % HA-Cat (e.g., up to about 1.9 wt %, up to about 1.8 wt %, up to about 1.7 wt %, up to about 1.6 wt %, up to about 1.5 wt %, up to about 1.4 wt %, up to about 1.3 wt %, up to about 1.2 wt %, up to about 1.1 wt %, up to about 1 wt %, up to about 0.9 wt %, up to about 0.8 wt %, up to about 0.7 wt %, up to about 0.6 wt %, up to about 0.5 wt %, up to about 0.4 wt %, up to about 0.3 wt %, up to about 0.2 wt %, up to about 0.1 wt %, 0 wt %). In some aspects, the cell scaffold can include greater than 0 wt % HA-Cat (e.g., greater than about 0.1 wt %, greater than about 0.2 wt %, greater than about 0.3 wt %, greater than about 0.4 wt %, greater than about 0.5 wt %, greater than about 0.6 wt %, greater than about 0.7 wt %, greater than about 0.8 wt %, greater than about 0.9 wt %, greater than about 1 wt %, greater than about 1.1 wt %, greater than about 1.2 wt %, greater than about 1.3 wt %, greater than about 1.4 wt %, greater than about 1.5 wt %, greater than about 1.6 wt %, greater than about 1.7 wt %, greater than about 1.8 wt %, greater than about 1.9 wt %, greater than about 2 wt %).

It is considered that the cell scaffold can include an amount of HA-Cat ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the cell scaffold can include from 0 wt % to about 2 wt % HA-Cat (e.g., from about 0.1 wt % to about 1.9 wt %, from about 0.2 wt % to about 1.8 wt %, from about 0.3 wt % to about 1.7 wt %, from about 0.4 wt % to about 1.6 wt %, from about 0.5 wt % to about 1.5 wt %, from about 0.6 wt % to about 1.4 wt %, from about 0.7 wt % to about 1.3 wt %, from about 0.8 wt % to about 1.2 wt %, from about 0.9 wt % to about 1.1 wt %, from 0 wt % to about 1 wt %, from about 0.1 wt % to about 0.9 wt %, from about 0.2 wt % to about 0.8 wt %, from about 0.3 wt % to about 0.7 wt %, from about 0.4 wt % to about 0.6 wt %, from about 1 wt % to about 2 wt %, from about 1.1 wt % to about 1.9 wt %, from about 1.2 wt % to about 1.8 wt %, from about 1.3 wt % to about 1.7 wt %, from about 1.4 wt % to about 1.6 wt %).

In some aspects, the HAMA can have a molecular weight of at least about 50 kDa (e.g., at least about 100 kDa, at least about 150 kDa, at least about 200 kDa, at least about 250 kDa, at least about 300 kDa, at least about 350 kDa, at least about 400 kDa, at least about 450 kDa, at least about 500 kDa, at least about 550 kDa, at least about 600 kDa, at least about 650 kDa, at least about 700 kDa, at least about 750 kDa, at least about 800 kDa, at least about 850 kDa, at least about 900 kDa, at least about 950 kDa, at least about 1000 kDa, at least about 1100 kDa, at least about 1200 kDa, at least about 1300 kDa, at least about 1400 kDa, at least about 1500 kDa, at least about 1600 kDa, at least about 1700 kDa, at least about 1800 kDa, at least about 1900 kDa, at least about 2000 kDa). In some aspects, the HAMA can have a molecular weight of up to about 2000 kDa (e.g., up to about 1900 kDa, up to about 1800 kDa, up to about 1700 kDa, up to about 1600 kDa, up to about 1500 kDa, up to about 1400 kDa, up to about 1300 kDa, up to about 1200 kDa, up to about 1100 kDa, up to about 1000 kDa, up to about 950 kDa, up to about 900 kDa, up to about 850 kDa, up to about 800 kDa, up to about 750 kDa, up to about 700 kDa, up to about 650 kDa, up to about 600 kDa, up to about 550 kDa, up to about 500 kDa, up to about 450 kDa, up to about 400 kDa, up to about 350 kDa, up to about 300 kDa, up to about 250 kDa, up to about 200 kDa, up to about 150 kDa, up to about 100 kDa, up to about 50 kDa).

It is considered that the HAMA can have a molecular weight ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the HAMA can have a molecular weight of from about 50 kDa to about 2000 kDa (e.g., from about 100 kDa to about 1900 kDa, from about 150 kDa to about 1800 kDa, from about 200 kDa to about 1700 kDa, from about 250 kDa to about 1600 kDa, from about 300 kDa to about 1500 kDa, from about 350 kDa to about 1400 kDa, from about 400 kDa to about 1300 kDa, from about 450 kDa to about 1200 kDa, from about 500 kDa to about 1100 kDa, from about 550 kDa to about 1000 kDa, from about 600 kDa to about 950 kDa, from about 650 kDa to about 900 kDa, from about 700 kDa to about 850 kDa, from about 750 kDa to about 800 kDa, from about 50 kDa to about 800 kDa, from about 100 kDa to about 750 kDa, from about 150 kDa to about 700 kDa, from about 200 kDa to about 650 kDa, from about 250 kDa to about 600 kDa, from about 300 kDa to about 550 kDa, from about 350 kDa to about 500 kDa, from about 400 kDa to about 450 kDa, from about 750 kDa to about 2000 kDa, from about 800 kDa to about 1900 kDa, from about 850 kDa to about 1800 kDa, from about 900 kDa to about 1700 kDa, from about 950 kDa to about 1600 kDa, from about 1000 kDa to about 1500 kDa, from about 1100 kDa to about 1400 kDa, from about 1200 kDa to about 1300 kDa).

In some aspects, the HA-Cat can have a molecular weight of at least about 50 kDa (e.g., at least about 100 kDa, at least about 150 kDa, at least about 200 kDa, at least about 250 kDa, at least about 300 kDa, at least about 350 kDa, at least about 400 kDa, at least about 450 kDa, at least about 500 kDa, at least about 550 kDa, at least about 600 kDa, at least about 650 kDa, at least about 700 kDa, at least about 750 kDa, at least about 800 kDa, at least about 850 kDa, at least about 900 kDa, at least about 950 kDa, at least about 1000 kDa, at least about 1100 kDa, at least about 1200 kDa, at least about 1300 kDa, at least about 1400 kDa, at least about 1500 kDa, at least about 1600 kDa, at least about 1700 kDa, at least about 1800 kDa, at least about 1900 kDa, at least about 2000 kDa). In some aspects, the HA-Cat can have a molecular weight of up to about 2000 kDa (e.g., up to about 1900 kDa, up to about 1800 kDa, up to about 1700 kDa, up to about 1600 kDa, up to about 1500 kDa, up to about 1400 kDa, up to about 1300 kDa, up to about 1200 kDa, up to about 1100 kDa, up to about 1000 kDa, up to about 950 kDa, up to about 900 kDa, up to about 850 kDa, up to about 800 kDa, up to about 750 kDa, up to about 700 kDa, up to about 650 kDa, up to about 600 kDa, up to about 550 kDa, up to about 500 kDa, up to about 450 kDa, up to about 400 kDa, up to about 350 kDa, up to about 300 kDa, up to about 250 kDa, up to about 200 kDa, up to about 150 kDa, up to about 100 kDa, up to about 50 kDa).

It is considered that the HA-Cat can have a molecular weight ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the HA-Cat can have a molecular weight of from about 50 kDa to about 2000 kDa (e.g., from about 100 kDa to about 1900 kDa, from about 150 kDa to about 1800 kDa, from about 200 kDa to about 1700 kDa, from about 250 kDa to about 1600 kDa, from about 300 kDa to about 1500 kDa, from about 350 kDa to about 1400 kDa, from about 400 kDa to about 1300 kDa, from about 450 kDa to about 1200 kDa, from about 500 kDa to about 1100 kDa, from about 550 kDa to about 1000 kDa, from about 600 kDa to about 950 kDa, from about 650 kDa to about 900 kDa, from about 700 kDa to about 850 kDa, from about 750 kDa to about 800 kDa, from about 50 kDa to about 800 kDa, from about 100 kDa to about 750 kDa, from about 150 kDa to about 700 kDa, from about 200 kDa to about 650 kDa, from about 250 kDa to about 600 kDa, from about 300 kDa to about 550 kDa, from about 350 kDa to about 500 kDa, from about 400 kDa to about 450 kDa, from about 750 kDa to about 2000 kDa, from about 800 kDa to about 1900 kDa, from about 850 kDa to about 1800 kDa, from about 900 kDa to about 1700 kDa, from about 950 kDa to about 1600 kDa, from about 1000 kDa to about 1500 kDa, from about 1100 kDa to about 1400 kDa, from about 1200 kDa to about 1300 kDa).

In some aspects, the cell scaffold can further include a crosslinker including 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (NHS) and/or thiolated polyethylene glycol (PEG).

In some aspects, the crosslinker can include up to about 1 wt % 2 NHS (e.g., up to about 0.95 wt %, up to about 0.9 wt %, up to about 0.85 wt %, up to about 0.8 wt %, up to about 0.75 wt %, up to about 0.7 wt %, up to about 0.65 wt %, up to about 0.6 wt %, up to about 0.55 wt %, up to about 0.5 wt %, up to about 0.45 wt %, up to about 0.4 wt %, up to about 0.35 wt %, up to about 0.3 wt %, up to about 0.25 wt %, up to about 0.2 wt %, up to about 0.15 wt %, up to about 0.1 wt %, up to about 0.05 wt %, about 0 wt %). In some aspects, the crosslinker can include greater than 0 wt % NHS (e.g., greater than about 0.05 wt %, greater than about 0.1 wt %, greater than about 0.15 wt %, greater than about 0.2 wt %, greater than about 0.25 wt %, greater than about 0.3 wt %, greater than about 0.35 wt %, greater than about 0.4 wt %, greater than about 0.45 wt %, greater than about 0.5 wt %, greater than about 0.55 wt %, greater than about 0.6 wt %, greater than about 0.65 wt %, greater than about 0.7 wt %, greater than about 0.75 wt %, greater than about 0.8 wt %, greater than about 0.85 wt %, greater than about 0.9 wt %, greater than about 0.95 wt %, greater than about 1 wt %).

It is considered that the crosslinker can include an amount of NHS ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the crosslinker can include from 0 wt % to about 1 wt % NHS (e.g., from about 0.05 wt % to about 0.95 wt %, from about 0.1 wt % to about 0.9 wt %, from about 0.15 wt % to about 0.85 wt %, from about 0.2 wt % to about 0.8 wt %, from about 0.25 wt % to about 0.75 wt %, from about 0.3 wt % to about 0.7 wt %, from about 0.35 wt % to about 0.65 wt %, from about 0.4 wt % to about 0.6 wt %, from about 0.45 wt % to about 0.55 wt %, from 0 wt % to about 0.5 wt %, from about 0.05 wt % to about 0.45 wt %, from about 0.1 wt % to about 0.4 wt %, from about 0.15 wt % to about 0.35 wt %, from about 0.2 wt % to about 0.3 wt %, from about 0.5 wt % to about 1 wt %, from about 0.55 wt % to about 0.95 wt %, from about 0.6 wt % to about 0.9 wt %, from about 0.65 wt % to about 0.85 wt %, from about 0.7 wt % to about 0.8 wt %).

In some aspects, the crosslinker can include up to about 2 wt % thiolated PEG (e.g., up to about 1.9 wt %, up to about 1.8 wt %, up to about 1.7 wt %, up to about 1.6 wt %, up to about 1.5 wt %, up to about 1.4 wt %, up to about 1.3 wt %, up to about 1.2 wt %, up to about 1.1 wt %, up to about 1 wt %, up to about 0.9 wt %, up to about 0.8 wt %, up to about 0.7 wt %, up to about 0.6 wt %, up to about 0.5 wt %, up to about 0.4 wt %, up to about 0.3 wt %, up to about 0.2 wt %, up to about 0.1 wt %, 0 wt %). In some aspects, the crosslinker can include greater than 0 wt % thiolated PEG (e.g., greater than about 0.1 wt %, greater than about 0.2 wt %, greater than about 0.3 wt %, greater than about 0.4 wt %, greater than about 0.5 wt %, greater than about 0.6 wt %, greater than about 0.7 wt %, greater than about 0.8 wt %, greater than about 0.9 wt %, greater than about 1 wt %, greater than about 1.1 wt %, greater than about 1.2 wt %, greater than about 1.3 wt %, greater than about 1.4 wt %, greater than about 1.5 wt %, greater than about 1.6 wt %, greater than about 1.7 wt %, greater than about 1.8 wt %, greater than about 1.9 wt %, greater than about 2 wt %).

It is considered that the crosslinker can include an amount of thiolated PEG ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the crosslinker can include from 0 wt % to about 2 wt % thiolated PEG (e.g., from about 0.1 wt % to about 1.9 wt %, from about 0.2 wt % to about 1.8 wt %, from about 0.3 wt % to about 1.7 wt %, from about 0.4 wt % to about 1.6 wt %, from about 0.5 wt % to about 1.5 wt %, from about 0.6 wt % to about 1.4 wt %, from about 0.7 wt % to about 1.3 wt %, from about 0.8 wt % to about 1.2 wt %, from about 0.9 wt % to about 1.1 wt %, from 0 wt % to about 1 wt %, from about 0.1 wt % to about 0.9 wt %, from about 0.2 wt % to about 0.8 wt %, from about 0.3 wt % to about 0.7 wt %, from about 0.4 wt % to about 0.6 wt %, from about 1 wt % to about 2 wt %, from about 1.1 wt % to about 1.9 wt %, from about 1.2 wt % to about 1.8 wt %, from about 1.3 wt % to about 1.7 wt %, from about 1.4 wt % to about 1.6 wt %).

In some aspects, the cell scaffold can include at least about 5 mM Fe³⁺(e.g., at least about 5.5 mM, at least about 6 mM, at least about 6.5 mM, at least about 7 mM, at least about 7.5 mM, at least about 8 mM, at least about 8.5 mM, at least about 9 mM, at least about 9.5 mM, at least about 10 mM, at least about 10.5 mM, at least about 11 mM, at least about 11.5 mM, at least about 12 mM, at least about 12.5 mM, at least about 13 mM, at least about 13.5 mM, at least about 14 mM, at least about 14.5 mM, at least about 15 mM). In some aspects, the cell scaffold can include up to about 15 mM Fe³⁺ (e.g., up to about 14.5 mM, up to about 14 mM, up to about 13.5 mM, up to about 13 mM, up to about 12.5 mM, up to about 12 mM, up to about 11.5 mM, up to about 11 mM, up to about 10.5 mM, up to about 10 mM, up to about 9.5 mM, up to about 9 mM, up to about 8.5 mM, up to about 8 mM, up to about 7.5 mM, up to about 7 mM, up to about 6.5 mM, up to about 6 mM, up to about 5.5 mM, up to about 5 mM).

It is considered that the cell scaffold can include an amount of Fe³⁺ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the cell scaffold can include from about 5 mM to about 15 mM Fe³⁺ (e.g., from about 5.5 mM to about 14.5 mM, from about 6 mM to about 14 mM, from about 6.5 mM to about 13.5 mM, from about 7 mM to about 13 mM, from about 7.5 mM to about 12.5 mM, from about 8 mM to about 12 mM, from about 8.5 mM to about 11.5 mM, from about 9 mM to about 11 mM, from about 9.5 mM to about 10.5 mM, from about 5 mM to about 10 mM, from about 5.5 mM to about 9.5 mM, from about 6 mM to about 9 mM, from about 6.5 mM to about 8.5 mM, from about 7 mM to about 8 mM).

In some aspects, the cell scaffold can have a damping factor (tan δ) of at least about 0.03 (e.g., at least about 0.04, at least about 0.05, at least about 0.06, at least about 0.07, at least about 0.08, at least about 0.09, at least about 0.1, at least about 0.11, at least about 0.12, at least about 0.13, at least about 0.14, at least about 0.15, at least about 0.16, at least about 0.17, at least about 0.18, at least about 0.19, at least about 0.2, at least about 0.21, at least about 0.22, at least about 0.23, at least about 0.24, at least about 0.25, at least about 0.26, at least about 0.27, at least about 0.28, at least about 0.29, at least about 0.3). In some aspects, the cell scaffold can have a damping factor (tan δ) of up to about 0.3 (e.g., up to about 0.29, up to about 0.28, up to about 0.27, up to about 0.26, up to about 0.25, up to about 0.24, up to about 0.23, up to about 0.22, up to about 0.21, up to about 0.2, up to about 0.19, up to about 0.18, up to about 0.17, up to about 0.16, up to about 0.15, up to about 0.14, up to about 0.13, up to about 0.12, up to about 0.11, up to about 0.1, up to about 0.09, up to about 0.08, up to about 0.07, up to about 0.06, up to about 0.05, up to about 0.04, up to about 0.03).

It is considered that the cell scaffold can have a damping factor (tan δ) ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the cell scaffold can have a damping factor (tan δ) of from about 0.03 to about 0.3 (e.g., from about 0.04 to about 0.29, from about 0.05 to about 0.28, from about 0.06 to about 0.27, from about 0.07 to about 0.26, from about 0.08 to about 0.25, from about 0.09 to about 0.24, from about 0.1 to about 0.23, from about 0.11 to about 0.22, from about 0.12 to about 0.21, from about 0.13 to about 0.2, from about 0.14 to about 0.19, from about 0.15 to about 0.18, from about 0.16 to about 0.17, from about 0.03 to about 0.17, from about 0.04 to about 0.16, from about 0.05 to about 0.15, from about 0.06 to about 0.14, from about 0.07 to about 0.13, from about 0.08 to about 0.12, from about 0.09 to about 0.11, from about 0.16 to about 0.3, from about 0.17 to about 0.29, from about 0.18 to about 0.28, from about 0.19 to about 0.27, from about 0.2 to about 0.26, from about 0.21 to about 0.25, from about 0.22 to about 0.24).

In some aspects, the cell scaffold can have a compression modulus (E) of at least about 250 Pa (e.g., up to about 300 Pa, up to about 350 Pa, up to about 400 Pa, up to about 450 Pa, up to about 500 Pa, up to about 600 Pa, up to about 700 Pa, up to about 800 Pa, up to about 900 Pa, up to about 1000 Pa, up to about 1200 Pa, up to about 1400 Pa, up to about 1600 Pa, up to about 1800 Pa, up to about 2000 Pa, up to about 2200 Pa, up to about 2400 Pa, up to about 2600 Pa, up to about 2800 Pa, up to about 3000 Pa, up to about 3500 Pa, up to about 4000 Pa, up to about 4500 Pa, up to about 5000 Pa, up to about 5500 Pa, up to about 6000 Pa, up to about 6500 Pa, up to about 7000 Pa, up to about 7500 Pa, up to about 8000 Pa, up to about 8500 Pa, up to about 9000 Pa, up to about 9500 Pa, up to about 10,000 Pa). In some aspects, the cell scaffold can have a compression modulus (E) of up to about 10,000 Pa (e.g., up to about 9500 Pa, up to about 9000 Pa, up to about 8500 Pa, up to about 8000 Pa, up to about 7500 Pa, up to about 7000 Pa, up to about 6500 Pa, up to about 6000 Pa, up to about 5500 Pa, up to about 5000 Pa, up to about 4500 Pa, up to about 4000 Pa, up to about 3500 Pa, up to about 3000 Pa, up to about 2800 Pa, up to about 2600 Pa, up to about 2400 Pa, up to about 2200 Pa, up to about 2000 Pa, up to about 1800 Pa, up to about 1600 Pa, up to about 1400 Pa, up to about 1200 Pa, up to about 1000 Pa, up to about 900 Pa, up to about 800 Pa, up to about 700 Pa, up to about 600 Pa, up to about 500 Pa, up to about 450 Pa, up to about 400 Pa, up to about 350 Pa, up to about 300 Pa, up to about 250 Pa).

It is considered that the cell scaffold can have a compression modulus (E) ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the cell scaffold can have a compression modulus (E) of from about 250 Pa to about 10,000 Pa (e.g., from about 300 Pa to about 9500 Pa, from about 350 Pa to about 9000 Pa, from about 400 Pa to about 8500 Pa, from about 450 Pa to about 8000 Pa, from about 500 Pa to about 7500 Pa, from about 600 Pa to about 7000 Pa, from about 700 Pa to about 6500 Pa, from about 800 Pa to about 6000 Pa, from about 900 Pa to about 5500 Pa, from about 1000 Pa to about 5000 Pa, from about 1200 Pa to about 4500 Pa, from about 1400 Pa to about 4000 Pa, from about 1600 Pa to about 3500 Pa, from about 1800 Pa to about 3000 Pa, from about 2000 Pa to about 2800 Pa, from about 2200 Pa to about 2600 Pa, from about 250 Pa to about 2400 Pa, from about 300 Pa to about 2200 Pa, from about 350 Pa to about 2000 Pa, from about 400 Pa to about 1800 Pa, from about 450 Pa to about 1600 Pa, from about 500 Pa to about 1400 Pa, from about 600 Pa to about 1200 Pa, from about 700 Pa to about 1000 Pa, from about 800 Pa to about 900 Pa, from about 2200 Pa to about 10,000 Pa, from about 2400 Pa to about 9500 Pa, from about 2600 Pa to about 9000 Pa, from about 2800 Pa to about 8500 Pa, from about 3000 Pa to about 8000 Pa, from about 3500 Pa to about 7500 Pa, from about 4000 Pa to about 7000 Pa, from about 4500 Pa to about 6500 Pa, from about 5000 Pa to about 6000 Pa).

In some aspects, the cell scaffold can have a stress relaxation time of at least about 10 seconds (e.g., at least about 20 seconds, at least about 30 seconds, at least about 60 seconds, at least about 90 seconds, at least about 120 seconds, at least about 150 seconds, at least about 180 seconds, at least about 210 seconds, at least about 240 seconds, at least about 270 seconds, at least about 300 seconds, at least at least about 350 seconds, at least about 400 seconds, at least about 450 seconds, at least about 500 seconds, at least about 550 seconds, at least about 600 seconds, at least about 650 seconds, at least about 700 seconds, at least about 750 seconds, at least about 800 seconds, at least about 850 seconds, at least about 900 seconds, at least about 950 seconds, at least about 1000 seconds). In some aspects, the cell scaffold can have a stress relaxation time of up to about 1000 seconds (e.g., up to about 950 seconds, up to about 900 seconds, up to about 850 seconds, up to about 800 seconds, up to about 750 seconds, up to about 700 seconds, up to about 650 seconds, up to about 600 seconds, up to about 550 seconds, up to about 500 seconds, up to about 450 seconds, up to about 400 seconds, up to about 350 seconds, up to about 300 seconds, up to about 270 seconds, up to about 240 seconds, up to about 210 seconds, up to about 180 seconds, up to about 150 seconds, up to about 120 seconds, up to about 90 seconds, up to about 60 seconds, up to about 30 seconds, up to about 20 seconds, up to about 10 seconds).

It is considered that the cell scaffold can have a stress relaxation time ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the cell scaffold can have a stress relaxation time of from about 10 seconds to about 1000 seconds (e.g., from about 20 seconds to about 950 seconds, from about 30 seconds to about 900 seconds, from about 60 seconds to about 850 seconds, from about 90 seconds to about 800 seconds, from about 120 seconds to about 750 seconds, from about 150 seconds to about 700 seconds, from about 180 seconds to about 650 seconds, from about 210 seconds to about 600 seconds, from about 240 seconds to about 550 seconds, from about 270 seconds to about 500 seconds, from about 300 seconds to about 450 seconds, from about 350 seconds to about 400 seconds, from about 10 seconds to about 400 seconds, from about 20 seconds to about 350 seconds, from about 30 seconds to about 300 seconds, from about 60 seconds to about 270 seconds, from about 90 seconds to about 240 seconds, from about 120 seconds to about 210 seconds, from about 150 seconds to about 180 seconds, from about 350 seconds to about 1000 seconds, from about 400 seconds to about 950 seconds, from about 450 seconds to about 900 seconds, from about 500 seconds to about 850 seconds, from about 550 seconds to about 800 seconds, from about 600 seconds to about 750 seconds, from about 650 seconds to about 700 seconds).

In some aspects, the spinal cord spheroid or organoid or a fragment thereof may be human. In some aspects, the spinal cord spheroid or organoid or a fragment thereof can be formed by culturing induced pluripotent stem cells (iPSCs) on the cell scaffold.

In some aspects, the one or more ventral markers can include SOX2, FOXA2, LHX3, NKX2.2, and/or OLIG2; the one or more dorsal markers can include PAX7, LHX3, LMX1, LHX9, and/or BRN3; and the one or more interneuron markers can include DBX1, DBX2, and or PAX6.

In some aspects, compared to a reference spinal cord spheroid or organoid or fragment thereof not cultured on the cell scaffold, the spinal cord spheroid or organoid or fragment thereof can include at least about 1.5-fold greater expression of the one or more ventral markers (e.g., at least about 1.6-fold greater, at least about 1.7-fold greater, at least about 1.8-fold greater, at least about 1.9-fold greater, at least about 2-fold greater, at least about 2.1-fold greater, at least about 2.2-fold greater, at least about 2.3-fold greater, at least about 2.4-fold greater, at least about 2.5-fold greater).

In some aspects, compared to a reference spinal cord spheroid or organoid or fragment thereof not cultured on the cell scaffold, the spinal cord spheroid or organoid or fragment thereof can include at least about 2-fold greater expression of the one or more dorsal markers (e.g., at least about 2.1-fold greater, at least about 2.2-fold greater, at least about 2.3-fold greater, at least about 2.4-fold greater, at least about 2.5-fold greater, at least about 2.6-fold greater, at least about 2.7-fold greater, at least about 2.8-fold greater, at least about 2.9-fold greater, at least about 3-fold greater, at least about 3.1-fold greater, at least about 3.2-fold greater, at least about 3.3-fold greater, at least about 3.4-fold greater, at least about 3.5-fold greater, at least about 3.6-fold greater, at least about 3.7-fold greater, at least about 3.8-fold greater, at least about 3.9-fold greater, at least about 4-fold greater).

In some aspects, compared to a reference spinal cord spheroid or organoid or fragment thereof not cultured on the cell scaffold, the spinal cord spheroid or organoid or fragment thereof can include at least about 2-fold greater expression of the one or more interneuron markers (e.g., at least about 2.1-fold greater, at least about 2.2-fold greater, at least about 2.3-fold greater, at least about 2.4-fold greater, at least about 2.5-fold greater, at least about 2.6-fold greater, at least about 2.7-fold greater, at least about 2.8-fold greater, at least about 2.9-fold greater, at least about 3-fold greater).

Methods

In one aspect, provided is a method of modeling a spinal cord, the method including culturing a plurality of induced pluripotent stem cells on a cell scaffold to form a spinal cord spheroid or organoid or a fragment thereof, the cell scaffold including methacrylated hyaluronic acid (HAMA) and dopamine-modified hyaluronic acid (HA-Cat); wherein the method causes the spinal cord spheroid or organoid or a fragment thereof to express one or more ventral markers, dorsal markers, and/or interneuron markers.

In another aspect, provided is a method of modeling a blood-spinal cord barrier, the method including: a) culturing a plurality of induced pluripotent stem cells on a cell scaffold to form a spinal cord spheroid or organoid or a fragment thereof, the cell scaffold including methacrylated hyaluronic acid (HAMA) and dopamine-modified hyaluronic acid (HA-Cat); and b) co-culturing the spinal cord spheroid or organoid or a fragment thereof with a blood vessel spheroid or organoid or a fragment thereof; wherein step a) causes the spinal cord spheroid or organoid or a fragment thereof to express one or more ventral markers, dorsal markers, and/or interneuron markers.

Two example protocols for culturing the induced pluripotent stem cells to form the spinal cord spheroid or organoid or fragment thereof are described below. In one example, at day 0, the single induced pluripotent stem cells (i.e., iPSCs or hiPSCS) are seeded in 100 μL of DMEM/F12 plus N2B27 medium with 10 M Y-27632 in each well of a U-bottom low attachment 96-well plate at a density of 15,000 cells/well for hiPSC self-aggregation. At day 1, the cells are fed with N2B27 medium containing 10 μM Y-27632, 4 μM CHIR99021, and 0.5 μM LDN193189. For ventral spinal cord organoid differentiation, RA activators (retinoic acid) and SHH activators (purmorphamine) are added to the culture media at various stages for promoting ventral spinal cord patterning. At day 3, the self-assembled spheroids are transferred into hydrogels, which are layered on top and beneath the spheroids, and the neural induction medium containing 1 μM RA is added for generating ventral patterning. At day 10, the N2B27 medium is changed to 1 μM retinoic acid and 1 μM Purmorphamine. The medium is changed every other day. On day 18 and onwards, the medium is changed to N2B27 media supplemented with 10 ng/mL BDNF.

In another example, dissociated small clumps of induced pluripotent stem cells are seeded into the U-bottom low attachment 96-well plates at density of 15,000 cells/well for hiPSC self-aggregation with 100 μL neural induction medium with 10 μM Y-27632. After two days culture, the medium is changed to N2B27 medium with 10 μM SB431542 and 2 μM CHIR99021 for 3 days with daily medium change. At day 3, the self-assembled spheroids are transferred into hydrogels. At day 5, the medium is changed to N2B27 with 20 ng/mL bFGF. The spheroids are fed daily for four days and begin forming a neuroepithelial (NE) structure at the peripheral surface of the organoid. On day 9, the spheroids are cultured in NIM containing 0.1 μM RA without bFGF for 8 days, inducing neural plate morphogenesis in NEs to form the neural tube. The medium is changed every other day. For organoid maturation, the spheroids are grown in a 1:1 mixture of DMEM/F-12 and neurobasal medium (Life Technologies, 21103-049; the medium contained 0.5% N2, 2% B27, 0.5% NEAA, 1% P/S, 0.1% β-mercaptoethanol, 1% GlutaMAX, and 0.1 μM RA. The medium is changed every 3-5 days.

In some aspects, the induced pluripotent stem cells can include healthy cells. In other aspects, the induced pluripotent stem cells can include diseased or abnormal cells. For example, in some such aspects, the induced pluripotent stem cells can be derived from a subject having a neurodevelopmental disorder, a neurodegenerative disease or disorder, a neurological disease or disorder, or a cancer (e.g., a brain cancer or spinal cancer).

In some aspects, the blood vessel spheroid or organoid or a fragment thereof can include healthy cells. In other aspects, the blood vessel spheroid or organoid or fragment thereof can include diseased or abnormal cells. For example, in some such aspects, the blood vessel spheroid or organoid or fragment thereof can be derived from a subject having an aneurysm or a vascular cancer (e.g., angiosarcoma).

In some aspects, the method can produce any of the disclosed spinal cord models or blood-spinal cord barrier models described above.

In yet another aspect, provided is a method of screening a therapeutic agent, the method including: a) culturing a plurality of induced pluripotent stem cells on a cell scaffold to form a spinal cord spheroid or organoid or a fragment thereof, the cell scaffold including methacrylated hyaluronic acid (HAMA) and dopamine-modified hyaluronic acid (HA-Cat); and b) administering the therapeutic agent to the spinal cord spheroid or organoid or fragment thereof.

In some aspects, the method can use any of the spinal cord models or blood-spinal cord barrier models described above.

In some aspects, the therapeutic agent can be targeted to treat and/or prevent a neurodevelopmental disorder, a neurodegenerative disease or disorder, a neurological disease or disorder, brain cancer, or spinal cancer.

EXAMPLES Example 1: Viscoelasticity of Hyaluronic Acid Hydrogels Regulates Human Pluripotent Stem Cell-Derived Spinal Cord Organoid Patterning and Vascularization

The 3D ventral spinal cord organoids have been generated using cell cycle inhibitor and recapitulated spinal neurogenesis as well as rostro-caudal patterns for modeling motor neuron disease [9]. To promote spinal cord patterning, using extracellular matrix (ECM) or scaffolds may provide a 3D signaling network to better pattern spinal cord organoids [10]. Additionally, inclusion of vital structure such as blood spinal cord barrier (BSCB) in the organoid is important for studying the dysfunction of spinal cord [11]. The BSCB serves as an interface responsible for facilitating the transport of nutrients between the bloodstream and the spinal cord [12]. Due to the analogous structure to the blood brain barrier, the endothelial cells are the most important components for spinal cord vascularization. Additionally, isogenic human blood vessel organoids (hBVOs) possess the capability to generate vascular structures and can be used to co-culture with human spinal cord organoids (hSCOs) to include BSCB structure in the organoids, through spheroid fusion and assembly as shown in previous studies [13, 14].

3D ECMs have a variety of effects on cellular process due to different characteristics. Elasticity or stiffness, nanotopography, and chemical functionalities of ECMs all have an influence on cell spreading, proliferation, migration, differentiation, and organoid formation [15-17]. Well-engineered ECMs can provide a proper microenvironment to regulate cellular behaviors including tissue regeneration due to specific biochemical and biophysical cues [18, 19]. In particular, the patterning of tissues or organoids can be tailored by 3D ECMs. 3D scaffold biomaterials especially hydrogels can be fabricated to mimic static mechanical properties of biological tissues and ECMs in the human body [20, 21]. Besides spatial mechanical properties, the viscoelasticity, or temporal (time-dependent) properties of hydrogels provides in-time cues for tissues/organoids to sense [22, 23] and dynamic stimulation to respond. The viscoelasticity of ECMs is a temporal parameter of the materials which can apply dynamic stimulation to the cells surrounded by ECMs. By regulating viscoelasticity in addition to mechanical properties such as the Young's modulus, ECMs provide both spatial and temporal factors for neural tissue morphogenesis [24]. Recently, the viscoelasticity of biomaterials (e.g., alginate) has been assessed to regulate cell proliferation, migration, and spreading [25, 26]. In addition, the effects of ECM viscoelasticity on the generation of embryoid body-like structure from hiPSCs were revealed [22]. Using alginate hydrogels with arginine-glycine-aspartate (RGD) ligands, the hiPSC morphogenesis in 3D culture showed that RGD density and stress relaxation time influenced cell viability, proliferation, apicobasal polarization, and lumen formation [22]. Nevertheless, the influence of hydrogel viscoelasticity on the cell behaviors is at the nascent stage, and the effect on the spinal cord organoid patterning has not yet been investigated.

In the human body, the major components of ECMs in the central nervous system are hyaluronans [27, 28]. Hyaluronic acid (HA) in the tissue fluid helps the tissue resist osmotic compression and absorb compressive force [29, 30]. Additionally, the network of HA is assembled by the existence of proteoglycans. The brain and spinal cord ECMs lack the fibrous components, such as collagens [31, 32]. In the brain, the entanglement of HA network is stabilized by specific connection between tenascins and proteoglycans [33]. Furthermore, HA can be used for wound healing, tissue maintenance, and inflammation [34-36]. The specific molecular weight of HA in different body parts could promote tissue remodeling and homeostasis [37, 38]. For example, HA has a remarkable hydration capacity, and lack of HA causes reduced extracellular space volume in the brain [39]. In the brain, the entangled network of HA needs to be stabilized through linkage with proteins and chondroitin sulfate proteoglycans [33, 40]. Therefore, HA-based ECMs can be designed with various modifications and compositions to provide specific biochemical and biomechanical properties [41-44].

Hence, this study fabricated different HA-based hydrogels for the generation and recapitulation of the patterning of spinal cord organoids. The static properties such as the stiffness of the hydrogels are important for regulating the behaviors of hiPSCs. However, dynamic properties, or time-dependent feature of the polymer also have important effects on the morphogenesis and lineage-specific differentiation of hiPSCs. Therefore, HA hydrogels with different stiffness and viscoelasticity were fabricated and characterized, based on covalent bond crosslinked methacrylated HA (HAMA). Then, hiPSCs were seeded into different hydrogels and induced for hSCO differentiation and patterning. hBVOs and hSCOs from different hydrogels were cocultured and characterized for vascularization of the organoids, which may lead to the generation of blood spinal cord barrier. Furthermore, dopamine modified HA (HA-Cat) with Fe³⁺coordinated crosslinked hydrogels were mixed with HAMA hydrogels to make dual network penetration (i.e., HAMA@HA-Cat) hydrogels. The dual network penetrated hydrogels also regulated hSCO patterning. Together, this study has significant implications on the role of viscoelastic properties of hydrogels in establishing human organoid model systems for disease modeling and drug screening.

Materials and Methods

Materials and reagents: The vendors and catalog numbers of key materials and reagents are provided: Sodium hyaluronate (HA-100 k, HA-200 k, HA-1M, Lifecore Biomedical, Inc), Dopamine hydrochloride (Sigma, H8502), methacrylic anhydride (Sigma, 276685), sodium hydroxide (Sigma, 221465), 1-ethyl-3-(−3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, D1601), N-hydroxysuccinimide (Thermo Scientific Chemicals, 157270250), poly ethylene glycol (PEG)-dithiol (Creative PEGWorks, PLS-612), 2-Hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (NHS) (Sigma, 410896), Rho-associated protein kinase (ROCK) inhibitor Y27632 (Sigma, Y0503), mTeSR Plus (STEMCELL Technologies Inc., 100-0276), LDN193189 hydrochloride (Sigma, SML0559), DMEM/F-12 (Gibco™, 12400024), B-27™ Supplement (50×) (Gibco™, 17504044), CHIR99021 (a Wnt signaling activator, Sigma, SML1046), retinoic acid (RA, Sigma), purmorphamine (a sonic hedgehog signaling activator, Sigma, SML0868), Recombinant human fibroblast growth factor (FGF)-basic (bFGF, Peprotech, 100-18C), N-2 Supplement (100×) (Gibco™, 17502048), Neurobasal™ Medium (Gibco™, 21103049), Human Endothelial Serum-free Medium (hESFM) (Gibco™, 11111044), β-mercaptoethanol (Gibco™ 21985023), MEM Non-Essential Amino Acids (NEAA) Solution (100×) (Gibco™, 11140050), GlutaMAX™ Supplement (Gibco™, 35050061), Proteinase K (Research Products International Corp, P502200.1), LIVE/DEAD™ Viability/Cytotoxicity Kit for mammalian cells (Invitrogen™ L3224), N2B27 media: 50% of DMEM/F12 mix with 50% Neurobasal Medium supplemented with 0.5% N2, 2% B27, 0.5% NEAA, 1% Penicillin/Streptomycin (P/S), 0.1% β-mercaptoethanol, and 1% GlutaMAX, Brain-derived neurotrophic factor (BDNF, Peprotech, 450-02), and Growth factor-reduced Matrigel (Corning, 354230).

Synthesis and characterization of HAMA and HA-Cat: For HAMA synthesis, methacrylation of HA was performed by adding dropwise 1.1 mL of MA at 1% (v/v) to 100 mL of 1% (w/v) HA solution in phosphate buffered saline (PBS), pH 7.4, at 4° C., under magnetic stirring for 24 h. The pH of the solution was kept between 8 and 10 with the addition of 5 N NaOH, until no further pH changes were detected, which indicated that the reaction was complete. The solution was dialyzed for 4 days with a 12-14 kDa membrane in deionized water at 4° C. Then, HAMA was frozen and lyophilized. The obtained powder material was stored at −20° C. until further use.

For catechol functionalization of HAMA, i.e., HA-Cat synthesis, HAMA was dissolved in 2-(N-Morpholino) ethanesulfonic acid (MES) buffer (pH=4.5). Next, 0.03 mol/L NHS, 0.03 mol/L EDC and 0.05 mg/mL dopamine were added to a bottle and stirred overnight to fully react. After synthesis, the derivatives of HA underwent dialysis in de-ionized water for three days to purify. Then the solutions were frozen and lyophilized. After synthesis, Hi-NMR (Bruker spectrometers B600, FSU-NMR Facility) was performed to characterize modification after synthesis.

Hama and Hama@HA-Cat Hydrogel Fabrication and Characterization:

Hydrogel fabrication: To obtain the covalently crosslinked HAMA gels, the HAMAs were photo-crosslinked with dithiol-PEG. A total of nine gels were synthesized. HAMA (three groups with molecular weight at 100 k, 200 k, and 1,000 k) was dissolved at 1%, 0.5%, and 0.25% (w/w) in PBS, respectively. The mixed polymer precursor in PBS was incubated at 37° C. with 0.1% (w/v) of NHS and 0.5% thiolated PEG and then cured with Dymax light shields model 5000 EC flood (intensity: 225 mW/cm²) for 30 seconds.

For fabrication of the HAMA@HA-Cat hydrogels (@ means that the hydrogel is a dual penetration network), 1% wt HAMA (three groups: 100 k, 200 k, and 1,000 k of molecular weight) and 1% wt HA-Cat (1,000 k) were mixed at a ratio of 1:1. Then, the mixed polymer precursor in PBS was incubated at 37° C. with 0.1% (w/v) of NHS and 0.5% thiolated PEG and then cured with Dymax light shields model 5000 EC flood (intensity: 225 mW/cm²) for 30 seconds. After the crosslinking, 200 μL of 40 mM FeCl₃aqueous solution was added to the hydrogels. The bulk hydrogels were cut into granular hydrogels for better reaction with FeCl₃solution during HA-Cat crosslinking.

Characterization of hydrogels: The static elastic properties of the hydrogels were measured via compression tests performed on an ARES-G2 Rheometer using a parallel plate geometry (d=25 mm) (TA Instruments, New Castle, DE, USA) and strain rate of 0.0000667 s4. Each gel composition was characterized with three specimens for at least three independent measurements.

Rheological characterization was also performed with an ARES-G2 Rheometer using the parallel plate geometry (d=25 mm, gap 0.5 mm). Oscillatory rheometery was conducted to measure the elastic and viscous modulus of the hydrogels. At first, parallel discs of 25 mm in diameter were placed on the rheometer and a 25 mm flat plate geometry was used to measure the samples across a strain sweep to find linear viscoelastic region (LVR) of the HAMA hydrogels with the parameter at 6.28 rad/s, 37° C. and within the range of strain at 0.1-100%. Then, the 0.5% strain was chosen for the frequency sweep to get the rheological properties of HAMA hydrogels. At least 3 samples (0.5 mm thick) for each gel composition were characterized.

The stress relaxation test was then performed in hydrogels. All samples were put between parallel discs of 25 mm in diameter and a gap of 1 mm. Next, the stress-relaxation behavior was quantified at 10% strain, with all tests lasting from 500 s to 3000 s for the samples to reach the plateau for the hydrogels. Then, the relaxation time data were regressed by Maxwell model to get the relaxation time (τ).

The morphology of the hydrogels was examined using scanning electron microscopy (SEM). The HAMA hydrogels were freeze-dried in a lyophilizer (Labconco Corporation, Kansas City, MO, USA) for 2 days. Then, the samples were taken out carefully, fixed onto a SEM stage with carbon tape, and coated with a 10 nm gold layer to better reveal the hydrogel morphology. Observations were made using a FEI Helios G4 UC multi-technique dual beam (electron and Ga ion) Field Emission Scanning Electron Microscope (FESEM) (Thermo Fisher Scientific, Pittsburgh, PA) under low-vacuum conditions.

hiPSC 2D and 3D cultures for biocompatibility evaluation: Undifferentiated human iPSK3 cells (human foreskin fibroblasts reprogrammed with plasmid DNA encoding reprogramming factors OCT4, NANOG, SOX2, and LIN28) were maintained on Growth Factor-reduced Matrigel-coated surface in mTeSR serum-free medium as described in previous publications [13, 14]. Prior to hiPSC seeding, the sterile HAMA precursor solutions were added into the wells of tissue culture plates and then the solutions were cured under UV for 30 s to form HAMA hydrogels. For 2D culture, the hiPSC suspension (˜1×10′ cells) was added at 100 μL into the wells of 96-well tissue culture plates coated with Matrigel (to ensure undifferentiated hiPSC attachment), on top of which was layered with different types of hydrogels. The cells were allowed to settle down into the hydrogels for 15 min. Then additional 100 μL of media were added to each well of a 96-well plate. For 3D culture, the hydrogels were fabricated in the wells of ultralow attachment (ULA) 96-well plates (to prevent cells from attaching to the surface of the culture plates). The dissociated hiPSCs were seeded into the hydrogels by placing two concentrated droplets (50 μL each) of cells into the hydrogels, for a final density of 1×10′ cells per gel. After 5 min, additional 100 μL media were added to each well. The cultures were maintained for 7 days and the cells were characterized by DNA assay for proliferation and Live/Dead assay for viability [45, 46].

Human spinal cord organoid differentiation in hydrogels: Two hSCO differentiation protocols were evaluated before the experiments using hydrogels [47, 48]. After comparison, the ventral hSCO differentiation protocol was chosen for this study. Briefly, undifferentiated hiPSCs were dissociated by Accutase for 5-7 min. At day 0, the single cells were seeded in 100 μL of DMEM/F12 plus N2B27 medium with 10 μM Y-27632 in each well of a U-bottom low attachment 96-well plate at a density of 15,000 cells/well for hiPSC self-aggregation. At day 1, the cells were fed with N2B27 medium containing 10 μM Y-27632, 4 μM CHIR99021, and 0.5 μM LDN193189. At day 3, the neural induction medium containing 1 μM RA was added for generating ventral patterning. The medium was changed every other day. At day 10, the spheroids were embedded into 15 μL concentrated Matrigel (1:3 dilution with neural induction medium) and incubated for one hour. Then, N2B27 media supplemented with 1 μM RA and 1 μM Purmorphamine were added to each well for neural patterning without disturbing Matrigel droplets. At day 14, the Matrigel-embedded organoids were transferred to the rocker [49, 50] or PBS Vertical Wheel bioreactor (PBS Biotech Inc., CA, USA) [51, 52] for further expansion and maturation. On day 18 and onwards, the medium was changed to N2B27 media supplemented with 10 ng/mL BDNF. To evaluate the influence of different types of hydrogels, single hiPSCs were seeded at a density at 15,000 cell/well into ULA 96-well plate. At day 3, the self-assembled spheroids were transferred into hydrogels, which were layered on top and beneath the spheroids, for further hSCO differentiation or co-culture with hBVOs.

The protocol for differentiating caudal hSCO: For the other hSCO differentiation protocol (i.e., hSCOB), dissociated small clumps of hiPSCs were seeding into the U-bottom low attachment 96-well plates at density of 15,000 cells/well for hiPSC self-aggregation with 100 μL neural induction medium with 1 μM Y-27632. After two days culture, the medium was changed to N2B27 medium with 10 μM SB431542 and 1 μM CHIR99021 for 3 days with daily medium change. At day 5, the medium was changed to N2B27 with 20 ng/mL bFGF. The spheroids were fed daily for four days and began forming a neuroepithelial (NE) structure at the peripheral surface of the organoid. On day 9, hSCOs were cultured in NIM containing 0.1 μM RA without bFGF for 8 days, inducing neural plate morphogenesis in NEs to form the neural tube. The medium was changed every other day. For organoid maturation, hSCOs were grown in a 1:1 mixture of DMEM/F-12 and neurobasal medium (Life Technologies, 21103-049; the medium contained 0.5% N2, 2% B27, 0.5% NEAA, 1% P/S, 0.1% β-mercaptoethanol, 1% GlutaMAX, and 0.1 μM RA. The medium was changed every 3-5 days.

Human blood vessel organoid differentiation: The hBVO generation was modified from previous publications [53, 54]. hiPSCs were seeded in the wells of ULA 96 well plate at a density at 10,000 cells/well in mTeSR plus supplemented with 10 μM ROCK inhibitor Y-27632. To initiate differentiation at day 0, cells were treated with 6 μM CHIR99021 (Selleckchem) in BVO1 medium: DMEM/F12 supplemented with 2% B27, 0.5% NEAA, 1% P/S, 0.1% 0-mercaptoethanol, and 1% GlutaMAX. The medium was changed every other day until day 6. At day 6, the medium was switched to BVO2 medium: hESFM supplemented with 20 ng/mL bFGF, 10 μM RA, and 2% B27. At day 9, the organoids were replated to tissue culture plates or continued to grow in hESFM with 2% B27 for long-term culture.

The vascular differentiation (vsc) protocol: For vascular differentiation of hiPSCs, Y27632 was added to the medium with cell density at 0.5×10⁶per well at day 0. Then, the Wnt activator CHIR99021 (12 μM, STEMCELL Technologies Inc.) was added to the culture medium at day 3. At day 5, 7, and 9, the media (DMEM/F12 with 2% B27) were changed to treat the cell aggregates with bone morphogenetic protein (BMP) 4 (Peprotech) (20 ng/mL), vascular endothelial growth factor (VEGF)-A (Peprotech) (20 ng/mL), and bFGF (20 ng/mL). At day 11, cells were switched to the medium containing VEGF-A (20 ng/mL), and bFGF (20 ng/mL), SB431542 (10 μM) to promote endothelial cell (EC) differentiation and suppress pericyte differentiation. At day 13, the cell aggregates were embedded in Matrigel and overlaid with differentiation medium containing 15% fetal bovine serum (FBS), 20 ng/mL VEGF-A, and 20 ng/mL FGF-2. Medium was changed every two or three days. At day 18, vascular networks were observed and analyzed. The derived cells were referred as iECs.

hSCO co-culture with hBVOs: Spheroid or organoid fusion methods were evaluated for hSCO vascularization by co-culturing with hBVOs. One 9-day hBVO and one 25-day hSCO were added to the same well of ULA 96-well plate and the organoid fusion occurred spontaneously. After a two-day fusion, the assembled organoid was embedded into Matrigel. Then the organoids were transferred to a low attachment 6-well plate on the rocker. For cell tracker labeling, culture media were removed, and CellTracker™ Red (1:1000 dilution, ThermoFisher Scientific, Waltham, MA, USA) solution was added. hBVOs were incubated with CellTracker™ Red solution at 37° C. for one hour. Then the staining solution was removed followed with washing. One CellTracker™ Red labeled hBVO and one hSCO were put next to each other in the same well of 96-well plate for 2 days. Finally, the assembly of the two organoids were imaged. All hSCOs from different HAMA hydrogels were extracted from hydrogels using blunt pipette tips, then they were co-cultured and assembled with hBVOs. The assembloids were characterized for the marker expression of both hSCOs and hBVOs.

Characterization of cell proliferation and biocompatibility in hydrogels: Cell proliferation was determined by DNA quantitation using Picogreen. The cells were harvested and lysed with 0.1 mg/mL proteinase K (Fisher Scientific, Pittsburgh, PA) at 50° C. overnight. The lysates (100 μL) were mixed with 100 μL of 0.5% Picogreen (Molecular Probes, Eugene, OR) in a 96-well plate. The plate was incubated for 5 min in the dark and then read on a fluorescent plate reader with 485ex/528em (BioRad Laboratories, Hercules, CA). The biocompatibility of the hiPSCs in different hydrogels were evaluated using LIVE/DEAD™ Viability/Cytotoxicity Kit (Invitrogen™, Waltham, MA). The organoids were harvested and then dissociated to single cells by Accutase for 20-40 min. Then, a cell suspension at 1×10⁶cells/mL was prepared. Next, 6 μL of 50 μM Calcein AM and 2 μL of 2 mM ethidium homodimer-1 (EthD-1) were added to each mL of cell suspension. The mixture of dye and cells was incubated at room temperature (RT) for 15 min. The stained cells were acquired with BD FACSCanto™ II flow cytometer (Becton Dickinson) and analyzed by FlowJo software. The cell only, live only, dead only, and live and dead samples were prepared for two-color flow cytometry compensation.

Histology sectioning and immunohistochemistry: The hSCOs were harvested and placed into 1.5 mL centrifuge tubes and fixed with 10% neutral buffered formalin for 24 hours. Then, the samples were dehydrated by series of ethanol solutions. Briefly, hSCOs were sequentially transferred to 70%, 75%, 80%, and 90% ethanol for 15 min each. Next, the samples were put into 95% ethanol for 60 min twice. Lastly, samples were submerged in 100% ethanol for 60 min twice. After dehydration, hSCOs were transferred into xylene for two 30-60 min intervals. Samples were incubated with 60° C. paraffin for 60 min twice and embedded with paraffin at ideal position during overnight cooling. After embedding in paraffin, the samples were sectioned by microtome at 6 μm for each slice. The slice was transferred to warm water and then dried on glass slides. Then, the sections were deparaffinized by immersing in Xylene for 3 min twice. Next, the slides were immersed into 100% ethanol for 3 min twice, 95% ethanol for 3 min, 70% ethanol for 3 min and then put under running cold tap water to rinse. The wet sections were transferred into 95° C. Sodium Citrate Buffer (10 mM Sodium Citrate, 0.05% Tween 20, pH 6.0) for 30 min and then washed under running cold tap water for 10 min. Immunocytochemistry analysis of hSCO markers were performed on the sections. Yes-associated protein (YAP) staining was also performed on the sections using a similar procedure to immunocytochemistry.

Immunocytochemistry of organoids: The hSCOs were directly replated to Matrigel (1:50) coated tissue culture plate. hBVOs were first dissociated by Accutase for 40 min, and then replated to Matrigel (1:50) coated tissue culture plate. Then, after a 3-day growth, both samples were fixed with 4% paraformaldehyde and permeabilized with cold methanol for staining intracellular markers. The samples were then blocked with 5% fetal bovine serum (FBS) and incubated with various mouse or rabbit primary antibodies (TABLE 1). Next, secondary antibodies were added in staining buffer (2% FBS in PBS). The cells were washed three times each for 5 min. The samples were then stained using Hoechst 33342 (ThermoFisher, 1:2,000) to label cell nuclei and afterwards washed with PBS overnight. Images of stained organoids were captured using a fluorescent microscope (Zeiss Axio Observer) or a Zeiss LSM 880 confocal microscope.

TABLE 1

A list of antibodies for target markers.

Cells	Primary Antibody	Origin/Isotype	Supplier/Cat#	Dilution

Neural cells	CHX10	Mouse monoclonal	Santa Cruz,	1:100
		IgG2a	sc-365519
	LHX3	Mouse monoclonal	Santa Cruz,	1:100
		IgG2a	sc-293411
	SOX2	Mouse monoclonal	Santa Cruz,	1:100
		IgG1	sc-365823
	NKX6.1	Mouse monoclonal	Santa Cruz,	1:100
		IgG2a	sc-130385
	NKX2.2	Mouse monoclonal	Santa Cruz,	1:100
		IgG2b	sc-398951
	HNF3β	Mouse monoclonal	Santa Cruz,	1:100
		IgG2a	sc-101060
	OLIG2	Mouse monoclonal	Santa Cruz,	1:100
		IgG1	sc-515947
	HB9	Goat polyclonal	Santa Cruz,	1:100
		IgG	sc-22542
	PAX7	Mouse monoclonal	Santa Cruz,	1:100
		IgG1	sc-81648
	Nestin	Mouse IgG₁	Sigma, N5413	1:100
	β-Tubulin III	Mouse monoclonal	Millipore,	1:100
		IgG₁	MAB1637
Blood vessel	CD31	Goat polyclonal	Santa Cruz,	1:200
cells		IgG	sc-1506
	VE-cadherin	Goat polyclonal	Santa Cruz,	1:200
		IgG	sc-6458
	ZO-1	Mouse IgG₁	Life Technologies,	1:100
			33-9100
General	YAP	Rabbit IgG	Santa Cruz,	1:100
			sc-15407
Secondary	Alexa 488, goat anti-	—	Life Technologies,	1:200
	mouse IgG₁		A-21121
	Alexa 594, goat anti-		Life Technologies,	1:200
	mouse IgG		A-11058
	Alexa 488, goat anti-		Life Technologies,	1:200
	mouse IgG_2a		A-21131
	Alexa 488, goat anti-		Life Technologies,	1:200
	mouse IgG_2b		A-21141
	Alexa 488, goat anti-		Life Technologies,	1:200
	rabbit IgG		A-11034

Image analysis of organoid morphology and YAP localization: To measure spheroid or organoid circularity and area during the experiments, phase-contrast images of hSCOs were taken with a microscope using a 4× and 10× objective every day up to day 18. These images were quantified with Image J software from National Institutes of Health (NIH). Briefly, the perimeter of each individual spheroid/organoid was drawn manually, and the enclosed area and circularity was measured. For YAP localization, the sections of stained organoids were imaged using a Zeiss LSM 880 confocal microscopy. YAP localization (nuclear or cytoplasmic) was analyzed using a quantification method through ImageJ as reported in a previous study [46].

Flow cytometry analysis for phenotypic marker expression: Briefly, the hSCOs and hBVOs were dissociated into single cells using Accutase and pipetting for 40 min. Then, 1×10⁶cells per sample were fixed with 4% paraformaldehyde and washed with staining buffer (2% FBS in PBS). The dissociated cells were permeabilized with 100% cold methanol for intracellular markers, blocked, and then incubated with primary antibodies against Chx10, LHX3, SOX2, NKX6.1, Nkx2.2, HNF30, OLIG2, H139, PAX7 followed by the corresponding secondary antibody (TABLE 1). The cells were acquired with BD FACSCanto™ II flow cytometer (Becton Dickinson) and analyzed against isotype controls using FlowJo software.

Reverse transcription-polymerase chain reaction (RT-PCR): Total RNA was isolated from different cell samples using the RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. The isolated RNA samples were further treated with DNA-Free RNA Kit (Zymo, Irvine, CA, USA) to remove genomic DNA contamination [55]. Reverse transcription was carried out according to the manufacturer's instructions using 2 ng of total mRNA, anchored oligo-dT primers (Operon, Huntsville, AL), and Superscript III (Invitrogen, Carlsbad, CA, USA). The software Oligo Explorer 1.2Primers (Genelink, Hawthorne, NY, USA) was used to design the real-time PCR primers specific for target genes (TABLE 2). For normalization of expression levels, β-actin was used as an endogenous control. Using SYBR1 Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA), real-time PCR reactions were performed on an ABI7500 instrument (Applied Biosystems). The amplification reactions were performed as follows: 2 min at 50° C., 10 min at 95° C., and 40 cycles of 95° C. for 15 sec and 55° C. for 30 sec, and 68° C. for 30 sec following with a melt curve analysis. The Ct values of the target genes were first normalized to the Ct values of the endogenous control P3-actin. The corrected Ct values were then compared to the experimental control. Fold changes in gene expression were calculated using the comparative Ct method: 2^−(Δ ^ttreatment ^−ΔC ^tcontrol ⁾to obtain the relative expression levels.

TABLE 2

Primer sequence for target genes.

	Forward primer	Reverse primer
Gene	5′ to 3′	5′ to 3′

GLUT-1	AGCAACTGTGTGGTCCCTACG	AAGGTCCGGCCTTTAGTCTCA
	(SEQ ID NO: 1)	(SEQ ID NO: 21)

BCRP	CAGGTGTGCGTCAGAATCATC	TCCAGGAGTGGTCAGATTCC
		TT
	(SEQ ID NO: 2)	(SEQ ID NO: 22)

PGP	ACCACTCTCCCACCTCCCTTA	TTTAGCTGGGCTGCGTTTACA
	(SEQ ID NO: 3)	(SEQ ID NO: 23)

Dbx1	AGCGAGACGACGTTTCTGAAG	TAGGGAAAGGCGAAGGTCTTG
	(SEQ ID NO: 4)	(SEQ ID NO: 24)

Dbx2	GGTATGGCCCACCCAGAGATA	CTGTGACACCACGGCTTTCTT
	(SEQ ID NO: 5)	(SEQ ID NO: 25)

FOXA2	CGGATCGAGGACAAGTGAGAG	GGTGGGGGTGTTATGGATTTC
	(SEQ ID NO: 6)	(SEQ ID NO: 26)

Lmx1a	GGGAACCCAGAATGAGTTGGT	CCAATGATGTCCCCAGAAATG
	(SEQ ID NO: 7)	(SEQ ID NO: 27)

NKX2.2	GCCTCTCCTTCTGAACCTTGG	ACATTAACGCTGGGACGGTTT
	(SEQ ID NO: 8)	(SEQ ID NO: 28)

BRN3	CGGTAGGACTTGGCTGTGAGA	TGTTCTGTTTTCGCCCAACAT
	(SEQ ID NO: 9)	(SEQ ID NO: 29)

LHX9	AGCCCTGCTTCTAGCCAAT	TGTTGTGAGGGCAGAGCACTA
	GTGCTCCGGACCATGAAA	CAAGATTTGTTCTCCCTGCA
	TCCTA	AA
	(SEQ ID NO: 10)	(SEQ ID NO: 30)

OLIG2	AAACTCCTCCACGTGCTTCCT	TGTTACACGGCAGACGCTACA
	(SEQ ID NO: 11)	(SEQ ID NO: 31)

VWF	CCTCAACTGCCACCAATGACT	GAACTGGCCCACAGGGTAGAT
	(SEQ ID NO: 12)	(SEQ ID NO: 32)

ZO1	CGGGACTGTTGGTATTGGCTA	CCCCCATTTACTGGCTGGTAT
	(SEQ ID NO: 13)	(SEQ ID NO: 33)

ISL1	GCCAGTCCAGAGAGACACGAC	GTCACTCTGCAAGGCGAAGTC
	(SEQ ID NO: 14)	(SEQ ID NO: 34)

OCLN	CCAATTGCTGCCACAAGAACT	ATTTCTCCAAGGTCCCACAGC
	(SEQ ID NO: 15)	(SEQ ID NO: 35)

CD31	AGTGTGACAAGCGTCATGGTG	AGGCTTTGGTGAGACCCACTT
	(SEQ ID NO: 16)	(SEQ ID NO: 36)

CDH5	TCGCTGTTGTCACATCTCAGG	AGTGGAAGATGCATGGGTGAC
	(SEQ ID NO: 17)	(SEQ ID NO: 37)

CLDN1	TGGGTTTCTTGCCTTAACCAG	AGAAAGCATCGGGCCATACTC
	(SEQ ID NO: 18)	(SEQ ID NO: 38)

GFAP	GATCTCTGCCTCAGTGCTCCA	GATATGCAGGAGGGTGGGTTT
	(SEQ ID NO: 19)	(SEQ ID NO: 39)

SELP	TAGCGATGAACTGCTCCAACC	CCATTCTCTTGGCATGCTGTT
	(SEQ ID NO: 20)	(SEQ ID NO: 40)

Whole-patch clamping for electrophysiology: Whole-cell patch clamp was used to record mature spinal cord spheroids cultured on small petri dish. The vessels were washed three times with extracellular recording solution containing 136 mM NaCl, 4 mM KCl, 2 mM MgCl, 10 mM HEPES, and 1 mM EGTA (312 mOsm, pH 7.39) and then were incubated in this solution at RT during recording. Glass electrodes (resistance 1-5 MQ) were filled with intracellular solution containing 130 mM KCl, 10 mM HEPES, and 5 mM EGTA (292 mOsm, pH 7.20). Cells were visualized under phase contrast with a Nikon Eclipse Ti-U inverted microscope with an attached DS-Qil monochrome digital camera. Recordings were made with an Axopatch 200B amplifier (Molecular Devices) and digitized with a Digidata 1440A system (Molecular Devices). Ionic currents were recorded under a voltage clamp protocol (−60 mV to 135 mV in 15 mV steps, 250 ms in duration).

Statistical Analysis: The differences were analyzed by independent t-test or one-way ANOVA followed by Tukey's multiple comparisons post hoc tests. The difference was considered statistically significant at p≤0.05 and all quantitative data are presented as mean±standard deviation.

Results

HA Hydrogel fabrication and characterizations: In FIG. 1A, the schematic illustrations demonstrate the fabrication process of HAMA and HAMA@HA-Cat hydrogels. The modification of HA with methylate group, catechol groups, and both groups were verified from the H¹NMR results (FIG. 2A). The double bond peaks introduced by the modification of MA appeared at 5.60 ppm and 6.04 ppm and the benzene ring peak introduced by the modification of dopamine appeared at 6.72 ppm, 7.10 ppm, and 7.13 ppm. Then the degrees of modification of 100 k, 200 k and 1,000 k HAMA are 50.4%, 50.0%, 45.4%, respectively.

HAMA hydrogels were fabricated with 3 different molecular weights and each sample was dissolved in PBS at 1%, 0.5%, and 0.25% (w/w) concentration (FIG. 2B). Different concentrations of HAMA could change the degree of crosslinking during gelation which leads to different mechanical properties [23]. Then, the mechanical and rheological properties were tested using a TA Ares-G2 (FIGS. 1B-1C, FIGS. 2C-2D, FIG. 3 , FIGS. 4A-4C). The storage modulus of the hydrogels decreased, and viscoelasticity increased with decrease in the mass fraction. The tan δ of the gels was between 0.044 and 0.154 for the selected groups (Gel 1-4). Then a group of compression modulus (E) was derived from compression test and was found to be in the range of 400 and 7,000 Pa. In TABLE 3, four types of hydrogels were selected from the 9 synthesized hydrogels, which can provide stiff-elastic (Gel 1), soft-elastic (Gel 2), stiff-viscoelastic (Gel 3), and soft-viscoelastic (Gel 4) hydrogel conditions. Usually, stress relaxation is used to evaluate the viscoelasticity of the polymer materials. Therefore, the stress relaxation test was also performed for the four HAMA hydrogels and the data were regressed with updated maxwell model (FIG. ED) [56]:

σ (t) = (σ_{0} - C) e^{\frac{- t}{τ}} + C (plateau)

where σ stands for stress (Pa), σ₀stands for initial stress (Pa), t stands for time (s), τ stands for relaxation time (s) and C is a constant relating to the crosslink of the polymer. The four stress relaxation times are 420 s, 660 s, 266 s, and 19 s, respectively. All relaxation times (τ) of the four samples are relatively short but significantly different. The degree of crosslinking of the hydrogels may be high with less fluid or dynamic part. The morphology of HAMA hydrogels is shown in SEM images, with visible porous structure (FIG. 5 ). These results indicate that the hydrogels with different viscoelastic properties, i.e., stiff-elastic (Gel 1), soft-elastic (Gel 2), stiff-viscoelastic (Gel 3), and soft-viscoelastic (Gel 4), can be fabricated.

TABLE 3

Properties of HAMA hydrogels with different molecular weights and concentrations.

	100 k	100 k	100 k	200 k	200 k	200 k	1,000 k	1,000 k	1,000 k
Parameters	1%	0.5%	0.25%	1%	0.5%	0.25%	1%	0.5%	0.25%

Tan δ	0.037	0.038	0.079	0.066	0.113	0.149	0.041	0.048	0.158
E(Pa)	5283 ±	1265 ±	605 ±	5050 ±	2624 ±	1208 ± 90	7456 ±	2213 ±	419 ± 127
	265	143	97	348	112		539	384
Stress	420.1	666.7	n/a	n/a	n/a	283.4	n/a	n/a	19.7
relaxation
time(s)
Category	stiff-	soft-	n/a	n/a	n/a	stiff-	n/a	n/a	soft-
	elastic	elastic				viscoelastic			viscoelastic
ID	Gel 1	Gel 2	n/a	n/a	n/a	Gel 3	n/a	n/a	Gel 4

Evaluation of hSCO derivation from hiPSCs: The schematic illustration of hSCO differentiation from hiPSCs reveals that differentiation was induced using LDN 193189 (inhibition of bone morphogenetic protein signaling), CHIR (Wnt activation), RA (retinoid activation), and Purmorphamine (Sonic Hedgehog signaling activation) (FIG. 6A) [47]. The ventral spinal cord organoids were generated and characterized for spinal cord markers of different regions, including dorsal, interneuron, and ventral markers (TABLE 4). The marker expression was compared with undifferentiated hiPSC aggregates (FIG. 7A). After 23 days of differentiation, the gene expression of ventral markers (SOX2, LHX3, NKX2.2, OLIG2) for the hSCO group was much higher than the hiPSC group, indicating the effective induction of hSCO lineage. PAX6 (a progenitor marker) had no difference between the hSCOs and the hiPSCs while ISL1 (a progenitor marker) and Nanog (a pluripotent gene) were higher for the hiPSC group. The current differentiation protocol (referred to as hSCOA) was furthered compared with a caudal hSCO differentiation protocol (referred to as hSCOB), which used SB431542 (inhibition of transforming growth factor signaling), CHIR, RA, and bFGF (FIG. 7B). hSCOA conditions showed higher LHX3, NKX2.2, OLIG2 expression, while PAX6 was comparable among the three groups. Based on these results, the hSCOA protocol was selected for the subsequent experiments. The hSCOs can be replated onto Matrigel-coated surface. Extended axons from the replated organoids were observed and the edges remained intact until day 44 (FIGS. 8A-8B). The hSCOs were maintained in the Vertical-wheel bioreactor for long-term culture until day 80, which showed larger organoids (˜2 mm) with the defined organoid edges.

TABLE 4

Summary of markers for hSCO patterning.

Early	SOX2	SOX2 is a neuroepithelial marker
progenitor	ISL1	A hindbrain progenitor marker
markers	NKX6.1	NKX 6.1 works in conjunction with other transcription
		factors to pattern the neural tube and regulate the
		differentiation of neuronal subtypes
Dorsal	LMX1a	it is involved in the development of the roof plate of the
markers		neural tube, which is important for the patterning of the
		dorsal spinal cord.
	LHX9	LHX9 is expressed in the dorsal spinal cord and is
		involved in the development of specific interneuron
		populations.
	BRN3	It is involved in the development of the nervous system,
		particularly in the differentiation of sensory neurons.
	LHX3	LHX3 is essential for the differentiation of specific classes
		of motor neurons, particularly those in the medial motor
		column, which innervate axial muscles
	PAX7	PAX 7 expressed in dorsal neural progenitors and plays a
		role in the development of dorsal spinal cord interneurons.
Interneuron	DBX1/DBX2	DBX1 is crucial for the specification of V0 and V1
markers		interneurons in the spinal cord, which are important for
		coordinating motor circuits.
		DBX2 is involved in the development of V2 interneurons
		in the spinal cord, which contribute to motor control and
		sensory processing.
	PAX6	It plays a role in the specification of dorsal spinal cord
		neurons, including certain sensory interneurons.
	CHX10	CHX 10 expressed in certain populations of neurons in the
		spinal cord, particularly in the V2a interneurons.
Ventral	NKX2.2	NKX2.2 is crucial for the development of ventral spinal
markers		cord neurons
	OLIG2	OLIG2 is critical for the development of motor neurons
		and oligodendrocyte progenitors in the ventral spinal cord
	FOXA2	It plays a crucial role in the development and
		differentiation of motor neurons in the ventral spinal cord.
	HNF3β	It is critical for the development of floor plate in the neural
		tube and for ventral patterning
Motor neurons	HB9	Mature motor neuronal marker

To verify the hSCO marker expression at the protein level, the day 18 organoids were replated for immunostaining (FIG. 6B and FIG. 9 ). Seven hSCO patterning markers were evaluated, and the expression of CHX10, LHX3, NKX6.1, HNF30, and OLIG2 was observed. In addition, neuroepithelial marker SOX2 had a high expression, indicating hSCO induction. Flow cytometry analysis was performed on day 25 samples to quantify the marker expression. Most ventral markers (except OLIG2) showed high expression (98.2% SOX2, 70.4% HNF30, and 48.0% NKX2.2). Dorsal markers PAX7 (39.2%) and LHX3 (77.0%) were also expressed (FIG. 6C). Of note, HB9, one of the motor neuron markers, was detected (FIG. 6D). To evaluate hSCO patterning, RT-PCR was performed to characterize gene expression of different functional regions of the spinal cord (FIG. 6E). For the ventral markers, NKX2.2 and OLIG2 had increased expression (3-4 fold) after one week of culture (day 25 vs. day 18, replated or not), when growth factors were withdrawn for maturation. FOXA2 showed no statistical difference. For interneuron markers, the expression of PAX6 was increased (˜3-fold), but not DBX1 and DBX2. The increased PAX6 expression may be due to the maturation of specific neural cells. For dorsal markers, BRN3 was expressed more (˜2-fold) after one week maturation, while the increase was not statistically significant for LMX1a and LHX9. These results indicate the effective hSCO derivation from hiPSCs for the investigation of hSCO patterning in the hydrogels and the extended differentiation time promotes hSCO maturation.

hSCO patterning in hydrogels with different stiffness and viscoelasticity: Next, for further hSCO differentiation within the hydrogels, the biocompatibility of the HAMA hydrogels was firstly investigated in 2D undifferentiated hiPSC culture and 3D undifferentiated hiPSC spheroids. The hiPSCs grew well when culturing with HAMA hydrogels during the 7-day period (FIG. 10A). By adding Matrigel, the adhesion of hiPSCs on 2D surface was improved. For 3D culture, the morphology (e.g., size) of hiPSC spheroids was similar with or without the addition of Matrigel. Then, hiPSCs were cultured in four different gels with a cell-only control. DNA assay was performed to evaluate cell growth and Live/Dead assay was performed to measure cell viability. The normalized DNA concentration shows that the proliferation of hiPSCs cultured with different HAMA hydrogels was comparable (FIG. 10B). The proliferation rates were lower than the cell-only condition, possibly because some of the hiPSCs were embedded into gels and did not proliferate much. For the Live/Dead assay, the five groups showed similar results with about 90% of live cells (FIG. 10C), which indicates that all the hydrogels have good biocompatibility for hiPSCs.

After the biocompatibility test, the spinal cord organoids (hSCOs) derived from hiPSCs were patterned in different HAMA hydrogels. Images of the formed spheroids in the four different hydrogels were taken over 18 days of differentiation (FIG. 10D and FIG. 11 ). The size of the spheroids increased significantly from day 5 (about 500-800 μm) to day 18 (about 1.5-1.8 mm). Image analysis was performed based on spheroid morphology to reveal if different HAMA hydrogels affect the spheroid size and shape. The quantitative summary of the diameter and circularity is shown in FIG. 10E. The diameters of all spheroids were similar (˜1.4 mm) for different hydrogel groups at day 15, however, they were different on the days prior to day 15, showing different growth kinetics of the spheroids. For example, the Gel 3 group started with the smaller spheroids but the spheroid size quickly increased to a size similar to the other groups. In addition, all spheroids in the HAMA hydrogels can freely grow without constriction from the hydrogels during the culture, which contributes to the size increase during the differentiation. The circularities of the spheroids all decreased over the course of differentiation (the closer to 1 the more circular). Only the spheroids in Gel 4 were less circular than the other conditions in the initial few days. These results indicate that the four types of HAMA hydrogels all support hSCO patterning.

After day 25, flow cytometry was performed to quantify hSCO marker expression at the protein level among different culture conditions (FIGS. 12A-12D and FIG. 13 ). The ventral markers of hSCOs were evaluated, and similar expression levels among different hydrogel groups were observed. The LHX3 (70-90%) and HNF30 (60-80%) had high expression while Nkx2.2 was expressed at 17-28%, OLIG2 was 10-16%, and CHX10 was 8-12%. Nkx6.1 expression was low around 2-7%. PAX7 showed large variations of 12-67%. The data from three different runs were normalized to the cell-only group and then combined together to make comparisons (FIGS. 12A-12B and FIG. 11 ). There were large variations among three different runs and no statistical difference was observed, which may be attributed to organoid-to-organoid variations [57].

Furthermore, RT-PCR was performed to evaluate patterning markers at the molecular level (FIG. 12C). For dorsal markers, Gel 1 reduced the expression of LMX1 and LHX9 while the highest expression was observed for the Gel 3 group. BRN3 expression was higher for the Gel 3 and Gel 4 groups in comparison to the Gel 1 group. For the interneuron marker expression, the presence of the hydrogels increased the expression of interneuron markers DBX1 and DBX2. Comparing with other gels, Gel 1 promoted higher expression of DBX1. PAX6 expression was higher for the Gel 3 and Gel 4 group in comparison to the Gel 1 group. For the ventral marker expression, Gel 3 promoted higher expression of FOXA2 and NKX2.2 in comparison to other conditions. There was no statistical difference among different conditions for OLIG2. Taken together, Gel 3 (stiff-viscoelastic) promoted dorsal and ventral marker expression and Gel 1 (stiff-elastic) promoted interneuron marker expression during hSCO patterning. These results indicate that the stiffer hydrogels are preferred for hSCO differentiation and the viscoelastic hydrogels promote regional hSCO patterning compared to the elastic hydrogels.

Electrophysiology was performed to show the functional properties of hSCOs (FIG. 12D). The electrophysiological properties of the outgrowth cells of the derived organoids were examined via patch clamping. The replated organoids displayed fast inward currents and long-lasting outward currents during voltage-clamp recording, suggesting the presence of functional voltage-gated Na⁺ and K⁺ channels, respectively.

Coculture of hSCOs from different hydrogels with hBVOs: The protocol of hBVO differentiation from hiPSCs was firstly evaluated for the marker expression of the endothelial cells (CD31, VWF) and tight junction (CDH5, CLDN1, ZO-1, OCLN, SELP, and GFAP) of the BSCB at different seeding densities (10,000, 20,000, and 30,000 cells/well in ULA 96-well plate) and replating (re) conditions (FIG. 14A). The vascular differentiation (vsc) from hiPSCs was also compared [46]. Only CDH5 showed different expression levels among different conditions. The 30 k, 30 k re, and 30 k vsc showed higher CDH5 expression than other densities. For the rest of the markers, the 10 k conditions had higher expression in general. In addition, the replated organoids did not show higher marker expression in comparison to organoids in suspension. Based on these RT-PCR results, the suspension culture and the seeding density of 10 k cells/well were selected for the generation of hBVOs. Then, the BMP4 and VEGF alone or in combination were tested as additional growth factors for hBVO generation (FIG. 14B). BMP4 significantly decreased the expression of tight junction and BSCB markers. Adding VEGF (with or without BMP4) did not significantly increase the marker expression in general. Therefore, the hBVO differentiation protocol without additional BMP4 and VEGF was used for subsequent experiments.

Afterwards, the assembly of hBVOs and hSCOs in the presence of HAMA hydrogels was performed (FIG. 15A). The hBVOs were labeled with CellTracker™ Red and transferred to a well containing one hSCO. The fusion of the hBVO with the hSCO was indicated by the red color inside the hSCO. With VEGF, the fusion rate was much faster than the other two conditions of no growth factor or with BMP4. After coculture for 3 weeks, the merged organoids (i.e., hSCO-hBVO) from five different conditions (i.e., Gel 1-4 and Gel-free) were harvested for RT-PCR analysis for the expression of spinal cord markers (FIG. 15B) or BSCB markers (FIG. 15C) [12]. For the spinal cord markers, DBX1 and LMX1a were higher for the Gel 2 group than the Gel 1 and Gel 3 groups, but comparable to the Gel 4 group. ISL1 had no difference among different hydrogel groups. The expression of OLIG2 of hSCOs derived from the Gel 4 group was higher than the Gel 1 group. NKX2.2 expression was the highest for the Gel 2 condition compared to the other groups. Taken together, the presence of hBVOs altered the influence of different hydrogels on spinal cord organoid patterning. Gel 2 (soft-elastic) promoted dorsal and interneuron markers as well as NKX2.2, while Gel 3 and 4 (viscoelastic) promoted ventral marker OLIG2 expression. These results indicate that the effects of viscoelastic properties of the hydrogels become more dominant than the stiffness effects.

For the BSCB markers in the fused hSCO-hBVO, the Gel 2 condition had the highest VWF (i) and OCLN (v) expression in comparison to the other groups (FIG. 15C). These markers are important for identifying the tight junction during coculture [58]. The tight junction protein ZO-1 (ii) was expressed higher in Gel 1 and Gel 3 groups when compared to the other conditions. The expression of glucose transporter 1 (GLUT-1) and efflux transporters, BCRP and PGP, was also determined. For PGP (iii), all the hSCO-hBVO conditions showed lower expression than the hBVO only group, due to the presence of hSCO cells. BRCP (iv) expression was comparable for all the conditions except for the Gel 4 group, which had lower expression. For GLUT-1 (vi), the Gel 3 group had the highest expression while Gel 4 had the lowest expression. Taken together, the BSCB markers were differentially affected by the viscoelastic properties of the HAMA hydrogels. Gel 2 (soft-elastic) promotes the tight junction and Gel 3 (stiff-viscoelastic) promotes the expression of glucose and efflux transporters.

HAMA-Cat (Fe³⁺) dynamic hydrogels for continuous hydrogel improvement: In addition to single covalent hydrogels that were investigated so far, the viscoelastic dual hydrogels with dynamic crosslinking bonds may also affect hSCO patterning. HAMA was crosslinked with covalent bonds and the entanglement of the chains provides the dynamic part that contributes to the viscoelastic behaviors of the hydrogels. In addition to modifying the covalent crosslinked HAMA hydrogels with dynamic crosslinked properties, dopamine, which has a catechol group, was grafted on the HA to synthesize HA-Cat (FIGS. 16A-16E). The catechol group can react with ferric ions (Fe³⁺) with coordination. The HAMA and HA-Cat polymers were mixed with the same concentration as the four HAMA hydrogels (100 k, 1%, 100 k 0.5%, 200 k 0.25%, 1000 k 0.25%), and the HAMA@HA-Cat hydrogels were fabricated which are referred as Gel 5, Gel 6, Gel 7, and Gel 8, respectively.

The mechanical properties of the four HAMA@HA-Cat hydrogels (Gel 5-8) were characterized. Using rheological tests, the shear modulus (FIG. 16A) and tan δ (FIG. 16B) of the four gels were measured. The modulus of Gel 8 was much lower than the other three groups and it had the highest tan δ of 0.3 in this study. Meanwhile, the compression modulus was determined, where Gel 5 and Gel 6 had similar compression moduli of ˜2,000 Pa and Gel 7 and Gel 8 had similar compression moduli of ˜400 Pa (FIG. 16C). Furthermore, the stress relaxation of the four hydrogels was evaluated, and the Maxwell model's regression was used to get the stress relaxation times of 373.0 s, 67.5 s, 94.6 s, and 19.4 s, respectively (FIG. 16D), all of which are less than those of the HAMA hydrogels. The hSCO patterning in the four HAMA@HA-Cat hydrogels was investigated (FIG. 16E). Gel 8 (the most viscoelastic HAMA@HA-Cat hydrogel) promoted the expression of dorsal marker BRN3A and interneuron markers DBX1 and DBX2 compared to other hydrogel conditions. Gel 6 promoted PAX6 expression. The ventral marker expression was not affected by different hydrogel properties. These results indicate that the viscoelasticity of dynamic hydrogels promotes hSCO patterning.

Mechanism of hydrogel effects on hSCO patterning: Considering the possible interplay between Hippo pathway and viscoelasticity of ECM, this study investigated the mechano-transduction mechanism by comparing the localization of YAP expression within the nuclei and cytoplasm (FIGS. 17A-17B) [46, 55, 59]. For this purpose, the hSCOs derived from different HAMA hydrogels were embedded in paraffin and sectioned into slices (6 D m) for better imaging of 3D structure. The YAP and Hoechst staining were observed in the hSCOs from different hydrogel groups (FIG. 17A). In addition, the localization of YAP in the nuclei and cytoplasm was compared through image analysis by ImageJ (FIG. 17B). The hSCOs from the more elastic hydrogel groups (Gel 1 and Gel 2) had lower YAP nuclear localization when comparing to the hSCOs from viscoelastic hydrogels (Gel 3 and 4). Of note, the hydrogels with similar tan □ but different modulus had no difference in YAP nuclear localization between each other, such as Gel land 2, Gel 3 and 4. These results indicate that more viscoelastic hydrogels could lead to more nuclear YAP localization.

Discussion

In this study, these findings provide a series of conclusions for 3D HAMA hydrogel microenvironments that influence the morphogenesis of hSCO and hSCO patterning under different viscoelasticity and stiffness of the static hydrogels as well as dynamic hydrogels. Using different concentrations and molecular weights of HA for crosslinking, the library of HAMA hydrogels with different mechanical property was established. For example, by using HA of different molecular weight with the same PEG-SH, the prorates can be manipulated. In addition, the ionic crosslinking mechanism and catechol chemistry were applied for hydrogel fabrication. Then, two groups of hydrogels with the similar modulus but different tan D (Gel 2 and Gel 3) were selected for mimicking different ECM properties. The effects of stress relaxation were tested to reveal the influence of the viscoelasticity of HAMA hydrogels on spinal cord organoid patterning. Another two groups of hydrogels were included to show the effects of hydrogels with the similar tan□ but different modulus (Gel 1 vs. Gel 2, Gel 3 vs. Ge14). At the early stage of lineage-specific hSCO differentiation, the culture kinetics of the size and circularity of organoids were affected by different hydrogels, e.g., Gel 3 group showed initial small size but later became similar to other conditions. For the differentiation and patterning of ventral hSCOs, the stiffness and viscoelasticity of the hydrogels had a greater influence on dorsal and interneuron marker expression but less on ventral markers.

It was only recently that the viscoelastic, or time-dependent properties of the extracellular environment, has been shown to have significant influences on cell and tissue behaviors [23]. To date, only a few reports focused on the influence of tunable viscoelastic property of the 3D matrices on the interactions between stem cells and the microenvironment [60]. Conventionally, the reports of culturing mesenchymal stem cells with 2D or 3D matrices are abundant, while the investigations of hiPSC-derived organoid generation in 3D viscoelastic matrices are still limited [61]. The matrix viscoelasticity has just recently been recognized as a key component for regulating stem cell organoid morphogenesis for tumor and intestine tissue modeling [22, 23]. Tunable stress relaxation (viscoelasticity), stiffness, and RGD ligands were shown to have significant effects on hiPSC apicobasal polarization and lumen formation [22]. Furthermore, the viscoelastic properties of HA hydrogels has been observed to promote human neural progenitor cell maturation in 2D culture, with faster stress-relaxation increasing neurite extension and decreasing metabolic activity [62]. Here, the HAMA and HAMA@HACat hydrogels were fabricated by simple synthetic methods that provided a range of different biophysical properties. Four categories of hydrogels were selected for patterning hiPSC-derived spinal cord organoids which provide specific microenvironments for hSCO differentiation. Furthermore, this study can maintain the similar stiffness and the same polymer composition during the process of hydrogel fabrication while still allowing for the generation of hydrogels with different viscoelastic properties. Therefore, the effects of the viscoelasticity of hydrogels on the hSCO morphogenesis and differentiation can be isolated from the stiffness effects, serving as the main variable of the biophysical cues. The influence of the porosity of the hydrogels was thought to be small and the difference in different hydrogels was small.

HA was chosen due to its abundance in human central nervous system and its ability to generate unique matrices to compare with other natural polymers, such as alginate, gelatin, etc. HA and its derivatives have been currently used as 3D matrices for cell/tissue culture, especially in 3D printing and as granular hydrogels [63-65]. The synthesis of HAMA hydrogels was based on a classical and simple method that can provide a series of hydrogels. As a result of the limited extent of grafting on the HA chains, the low degree of crosslinking leads to the entanglement of free HAMA chains and results in the viscoelastic properties of the HAMA hydrogels to some extent. Biochemical and biophysical properties are both important for hiPSC-derived organoids morphogenesis and patterning. Usually, the two factors have different effects on the regulation of fate decision of hiPSCs that are intrinsically sensitive to their biophysical and biochemical environment [66-68]. In addition, the spinal cord injury repair can be realized using the synthetic scaffolds with various biochemical and biophysical cues [69]. Once embedded within HA hydrogels, hiPSCs sense the signals from the matrix during embryoid body formation, differentiation induction, expansion, and hSCO patterning, which leads to different morphogenesis results. Furthermore, the Matrigel free condition provides low matrix affinity microenvironments for hiPSC spheroid formation and inhibits hiPSC expansion or attachment. Therefore, these HA hydrogels without any cell-attachment factors can provide biophysical signaling for organoid patterning in suspension with minimal influence of biochemical signaling. Furthermore, the analysis of YAP localization provides another angle to understand mechano-transduction mechanism of HAMA hydrogels with different modulus and viscoelasticity. This study found that the nuclear translocation of YAP increases for the hydrogels with faster stress relaxation for both values of elastic moduli (Gel 3 and Gel 4). These results are consistent with previous study using 2D substrate culture [70, 71].

Biophysical cues such as stiffness, nanotopography, and mechanical strain can regulate the fate of the hiPSCs, such as maintaining the pluripotency or inducing differentiation. For example, substrate stiffness can influence neural induction and subtype specification of hiPSCs [72]. In addition, the topographic properties of the substrates can promote hiPSC differentiation into specific neural lineage [73]. In addition to stimulation by biophysical stimuli, directional growth and lineage-specific development of hiPSCs can be facilitated by biochemical factors. Therefore, the competition and synergistic effects between these two types of factors need to be investigated. Based on these findings, viscoelastic (i.e., high tan δ or shorter relaxation time) microenvironments promote dorsal or interneuron marker expression of hSCOs. Specifically, the stiffer hydrogels are preferred for hSCO differentiation than the softer hydrogels, and the viscoelastic hydrogels promote regional hSCO patterning compared to the elastic hydrogels. The growth factors that were added to the cultures were primarily for ventral organoid differentiation. The sonic hedgehog activator and RA are the two key factors for ventral patterning of spinal cord organoids. Comparing the ventral markers of different hSCOs from different hydrogels showed no significant difference, showing that the ventral markers are mainly affected by the differentiation factors, not the biophysical properties of hydrogels.

Vascularization is essential to the growth, maturity, and function of organoids, as a crucial component in organoid development. The ability to remove waste materials and supply nutrients and oxygen to the cells inside the organoids depends on proper vascularization. Several techniques are used to promote vascularization in organoids, including: co-culture with ECs [74], embedding in Matrigel [75], microfluidic systems [76, 77], decellularized tissue scaffolds [78], and in vivo maturation [79]. In this study, co-culturing hSCOs with hBVOh for organoid fusion was used. The presence of hBVOs altered the influence of different hydrogels on spinal cord organoid patterning. The effects of viscoelastic properties of the hydrogels become more dominant than the stiffness effects. The presence of hSCOs also had effects on the expression of EC, tight junction, and BSCB markers in the presence of hydrogels. For example, soft-elastic hydrogels promoted the tight junction and stiff-viscoelastic hydrogels appeared to promote the expression of glucose and efflux transporters. However, the hBVOs in this study were not mature and the main assessment was based on the gene expression. The vascularization structure was not assessed, which may need the mature hBVOs and dynamic perfusion culture environment.

This study fabricated HAMA hydrogels with different modulus and viscoelasticity to regulate hSCO patterning and co-culture with hBVOs. The four hydrogels are mainly separated into 2 groups, the elastic and viscoelastic groups. After testing hSCO differentiation and biocompatibility of the four hydrogels, the morphogenesis of hSCOs were observed. The viscoelasticity of the hydrogels influenced the size and circularity. Then, by comparing the gene and protein expression of hSCOs with different hydrogels, the results reveal that the stiffer hydrogels are preferred for hSCO differentiation and the viscoelastic hydrogels promote regional hSCO patterning compared to the elastic hydrogels. By coculturing hSCOs and hBVOs, this study was able to create a fusion of the two organoids. In the presence of hBVOs, the effects of viscoelastic properties became more dominant than the stiffness effects. Soft-elastic hydrogels promoted the tight junction and stiff-viscoelastic hydrogels appeared to promote the expression of glucose and efflux transporters. The viscoelasticity of dynamic hydrogels was also found to promote hSCO patterning. Furthermore, by analysis of the localization of YAP, this study found that the nuclei localization increased in the faster relaxation hydrogel groups.

The following patents, applications and publications as listed below and throughout this document are hereby incorporated by reference in their entirety herein.

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Claims

What is claimed is:

1. A spinal cord model comprising:

a cell scaffold comprising methacrylated hyaluronic acid (HAMA) and dopamine-modified hyaluronic acid (HA-Cat); and

a spinal cord spheroid or organoid or a fragment thereof cultured on the cell scaffold and expressing one or more ventral markers, dorsal markers, and/or interneuron markers.

2. The spinal cord model of claim 1, wherein the cell scaffold comprises:

up to about 2 wt % HAMA; and

up to about 2 wt % HA-Cat.

3. The spinal cord model of claim 1, wherein:

the HAMA has a molecular weight of from about 50 kDa to about 2000 kDa; and

the HA-Cat has a molecular weight of from about 50 kDa to about 2000 kDa.

4. The spinal cord model of claim 1, wherein the cell scaffold further comprises a crosslinker comprising:

up to about 1 wt % 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone; and

up to about 2 wt % thiolated polyethylene glycol (PEG).

5. The spinal cord model of claim 1, wherein the cell scaffold further comprises from about 5 mM to about 15 mM Fe³⁺.

6. The spinal cord model of claim 1, wherein the cell scaffold has a damping factor (tan δ) of from about 0.03 to about 0.3.

7. The spinal cord model of claim 1, wherein the cell scaffold has a compression modulus (E) of from about 250 Pa to about 10,000 Pa.

8. The spinal cord model of claim 1, wherein the cell scaffold has a stress relaxation time of from about 10 seconds to about 1000 seconds.

9. The spinal cord model of claim 1, wherein the spinal cord spheroid or organoid or a fragment thereof is human.

10. The spinal cord model of claim 1, wherein the spinal cord spheroid or organoid or a fragment thereof is formed by culturing induced pluripotent stem cells (iPSCs) on the cell scaffold.

11. The spinal cord model of claim 1, wherein:

the one or more ventral markers comprise SOX2, FOXA2, LHX3, NKX2.2, and/or OLIG2;

the one or more dorsal markers comprise PAX7, LHX3, LMX1, LHX9, and/or BRN3; and

the one or more interneuron markers comprise DBX1, DBX2, and or PAX6.

12. The spinal cord model of claim 1, wherein, compared to a reference spinal cord spheroid or organoid or fragment thereof not cultured on the cell scaffold, the spinal cord spheroid or organoid or fragment thereof comprises:

at least about 1.5-fold greater expression of the one or more ventral markers;

at least about 2-fold greater expression of the one or more dorsal markers; and/or

at least about 2-fold greater expression of the one or more interneuron markers.

13. A blood-spinal cord barrier model comprising a spinal cord spheroid or organoid or a fragment thereof derived from the spinal cord model of claim 1 co-cultured with a blood vessel spheroid or organoid or a fragment thereof.